Highly sensitive method for detecting environmental insults

ABSTRACT

Subtle changes in environmental stress can now be detected and measured at sublethal levels as a generic response to environmental stress. The present invention provides a method to detect changes in the environmental stress level. The stress change is indicated as a change in the luminescence output of a genetically engineered microorganism. In the present invention, the luminescence gene complex is under the control of a stress inducible promoter.

CROSS-REFERENCE TO RELATED APPLICATIONS.

This application claims the benefit of the following prior-filed ornational or international applications. This application is a nationalfiling of PCT/US93/11527, Internt'l Filing Date 2 Dec. 1993, which inturn is a continuation-in-part of U.S. application Ser. No. 08/063,173,filed 14 May 1993, (now abandoned), which in turn is acontinuation-in-part of U.S. application Ser. No. 07/988,428, filed 4Dec. 1992, (now abandoned).

FIELD OF INVENTION

The invention relates to the detection of environmental insults atlevels below those necessary to compromise cell metabolism. Morespecifically the invention provides a transformed bacterial hostcontaining a DNA construct comprising a stress inducible promoteroperably connected to a reporter gene or gene complex such that thepresence of an environmental insult will induce the expression of thereporter genes. The preferred reporter genes are those that areresponsible for bacterial bioluminescence.

BACKGROUND

Increasing public concern and mounting government regulations haveprovided impetus for the development of environmental sensing systemscapable of detecting contaminants in soil and ground water. Highlysensitive and specific detection systems incorporating analytical toolssuch as Gas Chromatography and Mass spectrophotometry have been knownfor several years; however, these systems require expensive equipmentand skilled operators. Moreover, sample preparation and data analysis isoften cumbersome and time consuming. Toxicity assays involving livingorganisms such as Daphnia, used in the standard U.S. water toxicitytest, are simpler; however, these tests are non-specific and notparticularly rapid. Somewhat more rapid are cell based toxicity assaysthat incorporate a bacterial cell as the sensitive element. Thesesystems use bacterial cells as a reagent in a conventional automatedanalytical system. For example the RODTOX system (Central Kagaku.,Tokyo, Japan) is a batch assay that measures bacterial oxygenconsumption and was designed for use in sewage plants. Other bacteriabased systems such as the GBI TOXALARM system (Genossenschaft BerlinerIngenieuirkollective, Berlin, Germany) can measure the presence ofspecific chemicals. The GBI TOXALARM is known to be able to detect thepresence of as little as 0.1 ppm potassium cyanide in a sample. Thesedetection systems are useful, but are hampered by cumbersome and complexdetection systems. Recently, the phenomenon of bacterial bioluminescencehas been regarded as providing a simpler and more sensitive mode ofdetection in environmental sensing systems.

The phenomenon of bioluminescence first came under serious scientificscrutiny by Raphel Dubois in 1885 when he observed that the cell-freeextracts obtained from the luminescent beetle Pyrophrus and theluminescent clam Pholas gave a light emitting reaction in vitro whenmixed at room temperature. Since that time bioluminescent systems havebeen identified and examined in a myriad of different organismsincluding the common firefly, marine coelenterates, fish, terrestrialand freshwater worms, as well as bacterial, algal and fungal species.

Bacterial bioluminescence is phenomenon in which the products of 5structural genes (luxA, luxB, luxC, luxD and luxE) work in concert toproduce light. The luxD product generates a C14 fatty acid from aprecursor. The C14 fatty acid is activated in an ATP dependent reactionto an acyl-enzyme conjugate through the action of the luxE product whichcouples bacterial bioluminescence to the cellular energetic state. Theacyl-enzyme (luxE product) serves as a transfer agent, donating the acylgroup to the luxC product. The acyl-LuxC binary complex is then reducedin a reaction in which NADPH serves as an electron pair and proton donorreducing the acyl conjugate to the C14 aldehyde. This reaction couplesthe reducing power of the cell to bacterial light emission. The lightproduction reaction, catalyzed by luciferase (the product of luxA andluxB), generates light. The energy for light emission is provided by thealdehyde to fatty acid conversion and FMNH₂ oxidation, providing anothercouple between light production and the cellular energy state.

Recently, naturally bioluminescent organisms have been used as thesensitive element in toxicity tests. The MICROTOX system, (MicrobicsCorp., Carlsbad, Calif.) is an example. The MICROTOX system measures thenatural baseline luminescence of Photobacterium phosporeum and relatesthis to the hostility of the environment around the organism. Since thethree couples, ATP level, NADPH level and FMNH₂ level, between lightproduction and the central metabolic events of energy generation arenecessary for light production in Photobacterium phosporeum, any insultthat interferes with the availability or interaction of thesemetabolites will cause a decrease in the activity of thebioluminescence(lux) system and therefore a related decrease in lightproduction by the organism.

A main attribute of bioluminescent systems is that the decrease in lightproduction is rapid, occuring within minutes of exposure to an insult.Another key advantage of these systems is that light detection can beexquisitely sensitive (down to the level of single photons), and isreadily adaptable to portable field units. Furthermore, the logistics oflight detection precludes the necessity of having the detector contact awet, biological sample, which is a key weakness in competingtechnologies (such as ion-selective electrodes), where detector foulingand corrosion are responsible for significant down time.

Recent advances in recombinant DNA technology have made it possible toexpress the luciferase (lux) gene complex as heterologous gene products.This is generally accomplished by placing the lux structural genecomplex under the control of a host promoter. So, for example cDNAencoding firefly luciferase has been expressed in E. coli under thecontrol of the lacZ promoter. (Tatsumi et al., Biochem. Biophys Acta.,1131, (2), pp 161-165, (1992)), and the luxAB fusion gene has beenexpressed in Bacillus at levels comparable to those achievable in E.coli by placing it under the control of the powerful Pxyn promoter(Jacobs et al., Mol. Gen. Genet., 230(1-2), pp 251-256, (1991)).

Alternate systems to the MICROTOX system have been developed usingrecombinant genetics to transform bacteria to be the light emittingelement in the assay. Rychert et al. (Environ. Toxic. Water Qual., 6(4), pp 415-422, (1991)) have shown that recombinant E. coli harboringthe plasmid, pJE202, that contain the lux gene complex, was sensitive toZn²⁺, ethidium bromide, sodium pentachoropheate, Cu²⁺ and2,4-dichloropheoxyacetic acid. Response in this assay was registered bya decrease in baseline light emitted by the transformed E. coli.

Although the MICROTOX and similar systems are useful, their sensitivityis limited to detecting levels of insults that kill or cripple the cellmetabolically. To be detected by these systems, the insult must havereached a level high enough to either interfere with the centralmetabolism of the cell or to inactivate the Lux proteins.

Frequently it is necessary to be able to detect levels of insults atlevels below those needed to affect cell metabolism. Such is the case inwaste treatment facilities where lethal concentrations of pollutants caneradicate the useful microbial population, incurring significant costand plant down time. A preferred sensing system would be one that wouldbe able to detect the presence of insults at sublethal levels, before auseful microbial population could be harmed. Such an early warning couldbe used to trigger prompt remedial action to save the indigenousmicrobial population.

Recently, recombinant bacteria have been developed by fusing the luxstructural genes to chemically responsive bacterial promoters and thenplacing such chimeras in appropriate hosts. These recombinant bacteriaare sensor organisms that glow in response to specific stimuli. Anexample of this type of gene fusion is described by Burlage et al. (J.Bacteriol, 172 (9) pp 4749-4757 (1990)) where a DNA fragment fromplasmid NAH7 containing a promoter for the naphthalene degradationpathway was fused to the lux genes of Vibrio fischeri and used totransform a strain of Pseudomonas. The resulting transformant displayedan increase in light emission in the presence of naphthalene. Theinduction of bioluminescence was demonstrated to coincide withnaphthalene degradation by the transformed organism.

Another test system specifically responsive to mercury (Hg) is describedby H. Molders (EP 456667). Here, indicator bacterial strains areprovided (by vector mediated gene transfer) containing a met promoter,specifically inducible by Hg ions, fused to a bacterial luciferase (luxAB) genes complex which is responsible for bioluminescence. The testsystem of Molders relies on the induction of the met promoter by thepresence of mercury and the subsequent increase in light emission fromthe recombinant bacteria for the test results.

The methods of Burlage et al. and Molders offer several advantages overthe art in that they specifically detect a single insult by the methodof increased bioluminescence. These systems are useful for detecting thepresence of specific chemicals in an environmental sample but are poorindicators of general cell toxicity. The promoter used by Burlage isfunctional in the naphthalene degradative pathway and is placed in ahost that is able to use naphthalene as a carbon source. Hence thisdetection system is not associated with cell toxicity in any way.Similarly the mer-promoter of Molders is not indigenous to E. coli andtherefore is not a native indicator of toxicity in E. coli. A moregeneral test system for the primary detection of unknown insults wouldutilize a promoter specifically linked to cell toxicity or stress ratherthan one activated by one specific chemical.

Genes activated as a result of cellular stress provide an advantageousalternative strategy for the detection of environmental insults. Stressgenes are found in all cells and are defined as those genes activated asa result of any type of stress that might alter the normal cellularmetabolism. Environmental stresses often induce synthesis of anoverlapping set of proteins. The most well recognized class of stressgenes are the heat shock genes encoding a set of cellular proteinsthought to have roles in refolding, recycling and resynthesis ofproteins. The heat shock phenomenon was first described as a response toan increased temperature. Subsequent work has shown that exposure to avariety of stresses including phage infection, macrophage envelopment,as well as the presence of organic molecules and heavy metals can alsotrigger the heat shock response. The common theme of the inducing agentsmay be unfolding of some proteins within the cell. (LaRossa et al., Mol.Micriobiol., 5(3), pp 529-534, (1991)). Thus the response may integrateand report a wide range of environmental insults. VanBogelen et al. (J.Bacteriol, 169(1), pp 26-32, (1987)) have demonstrated that a variety ofchemicals are able to induce the heat shock genes in E. coli, includingCdCl₂, H₂ O₂, ethanol, puromycin and nalidixic acid. Blom et al. (Appl.Environ. Microbiol., 58(1), pp 331-334, (1992)) teach that the exposureof E. coli cultures to benzene, CdCl₂, chlorpyrivos, 2,4-dichloraniline,dioctylphtalate, hexachlorobenzene, pentachlorophenol,trichloroethylene, and tetrapropylbenzosulfonate leads to the inductionof up to 39 different stress proteins, as analyzed by two dimensionalgel electrophoresis.

Since the cell attempts to maintain a steady state, stress responses areactivated well below the minimal inhibitory concentration for anycondition that serves as a triggering factor. This fact would make theuse of stress responses in any environmental monitoring systemparticularly advantageous since detection of insults could beaccomplished before microbial cell death. Such a system would beextremely useful in waste treatment facilities where environmentalpollutants are often not detected until after the microbial populationhas been destroyed.

To date the induction of stress responses has been utilized in the areaof environmental testing with only moderate success. Koehler et al.(Arch. Environ. Contam. Toxicol., 22(3), 334-8, (1992)) describe a testsystem to assay the levels of HSP70 protein in various species ofmolluscs and slugs in response to the presence of heavy metals andpesticides. Although the system demonstrated increased levels of HSP70in response to the presence of Pb²⁺, the technique is cumbersome andlacks sensitivity. A more sophisticated technique described is bySaunders et al. (WO 9002947). The Saunders et al. technique involvesdetecting increased levels of HSP60 and HSP70 in organisms exposed topollutants in an aqueous environment.

Although stress responses have been demonstrated to be useful indetecting the presence of various environmental insults, it has yet tobe linked to a sensitive, easily detected reporter. A need exists,therefore for a highly sensitive biological test system employing afacile detection mechanism, able to detect a wide variety of insults atlevels well below those needed to kill microbial populations. It is theobject of the present invention to meet such a need. This invention isanticipated to have broad applicability. Potential uses includemonitoring of air and water quality, agrichemical and pharmaceuticaldesign, manufacturing and fermentation process control, processmonitoring and toxicity screening. These applications may benefit manyenterprises including the chemical, beverage, food and flavor,cosmetics, agricultural, environmental, regulatory and health careindustries.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting the presence of anenvironmental insult comprising the steps of: culturing a suitablebioluminescent detector organism capable of responding to the presenceof an environmental insult by a change in luminescence; exposing saiddetector organism to the presence of a sample suspected of containing anenvironmental insult; measuring the change in luminescence produced bythe detector organism; and correlating said change in luminescence withthe level of environmental insult present in said sample.

The present invention further provides a transformed bioluminescentbacterial host cell capable of a change in luminescence in response tothe presence of an environmental insult, wherein said host cell containsa heterologous DNA construct capable of being activated by the presenceof said insult.

Additionally the present invention provides a DNA fusion comprising afirst DNA fragment encoding a stress inducible promoter, operably andexpressibly connected to a second DNA fragment encoding the lux genecomplex.

BRIEF DESCRIPTION OF THE DRAWINGS AND BIOLOGICAL DEPOSITS

FIG. 1 is an illustration of the construction of plasmid pGrpELux.3 andpGrpELux.5.

FIG. 2 is an illustration of the construction of plasmid pRY006.

FIG. 3 is an illustration of the construction of plasmid pRY001 andpYR002.

FIG. 4 is a graphic representation of the increase in luminescence byTV1060 in response to the presence of ethanol.

FIG. 5 is a graphic representation of the increase in luminescence byTV1060 in response to the presence of increasing concentrations ofethanol.

FIG. 6a is a graphic representation of the increase in luminescence byWM1021 in response to the presence of varying concentrations of ethanol.

FIG. 6b is a graphic representation of the increase in luminescence byWM1026 in response to the presence of varying concentrations of ethanol.

FIG. 7 is a graphic representation of the relative sensitivities oftolC⁺ and tolC⁻ detector cells transformed with pGrpE.Lux.5 topentachlorophenol.

The following strains were deposited under the terms of the BudapestTreaty with the American Type Culture Collection (ATCC) (12301 PacklawnDrive, Rockville, Md. 20852, U.S.A.):

    ______________________________________                                        TV1060 (ATCC #69142).sup.+                                                                       WM1202 (ATCC #69313)*                                      TV1061 (ATCC #69315)*                                                                            WM1021 (ATCC #69141).sup.+                                 TV1076 (ATCC #69314)*                                                                            WM1026 (ATCC #69143).sup.+                                                    WM1302 (ATCC #69316)*                                      ______________________________________                                         (*deposited 13 May 1993)                                                      (.sup.+ deposited 3 December 1992)                                       

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used herein and should be referred to forclaim interpretation.

The terms "promoter" and "promoter region" refer to a sequence of DNA,usually upstream of (5' to) the protein coding sequence of a structuralgene, which controls the expression of the coding region by providingthe recognition for RNA polymerase and/or other factors required fortranscription to start at the correct site. Promoter sequences arenecessary but not always sufficient to drive the expression of the gene.

A "fragment" constitutes a fraction of the DNA sequence of theparticular region.

"Nucleic acid" refers to a molecule which can be single stranded ordouble stranded, composed of monomers (nucleotides) containing a sugar,phosphate and either a purine or pyrimidine. In bacteria and in higherplants, "deoxyribonucleic acid" (DNA) refers to the genetic materialwhile "ribonucleic acid" (RNA) is involved in the translation of theinformation from DNA into proteins.

"Regulation" and "regulate" refer to the modulation of gene expressioncontrolled by DNA sequence elements located primarily, but notexclusively upstream of (5' to) the transcription start of a gene.Regulation may result in an all or none response to a stimulation, or itmay result in variations in the level of gene expression.

The term "coding sequence" refers to that portion of a gene encoding aprotein, polypeptide, or a portion thereof, and excluding the regulatorysequences which drive the initiation of transcription. A coding sequencemay be one normally found in the cell or it may be one not normallyfound in a cellular location wherein, it is introduced, in which case itis termed a heterologous gene. The coding sequence may be a composite ofsegments derived from different sources, naturally occurring orsynthetic.

The term "construction" or "construct" refers to a plasmid, virus,autonomously replicating sequence, phage or nucleotide sequence, linearor circular, of a single- or double-stranded DNA or RNA, derived fromany source, in which a number of nucleotide sequences have been joinedor recombined into a unique construction which is capable of introducinga promoter fragment and DNA sequence for a selected gene product alongwith appropriate 3' untranslated sequence into a cell.

The term "transformation" refers to the acquisition of new genes in acell after the incorporation of nucleic acid.

The term, "operably linked" refers to the fusion of two fragments of DNAin a proper orientation and reading frame to be transcribed intofunctional RNA.

The term "expression" refers to the transcription and translation togene product from a gene coding for the sequence of the gene product. Inthe expression, a DNA chain coding for the sequence of gene product isfirst transcribed to a complimentary RNA which is often a messenger RNAand, then, the thus transcribed messenger RNA is translated into theabove-mentioned gene product if the gene product is a protein.

The term "translation initiation signal" refers to a unit of threenucleotides (codon) in a nucleic acid that specifies the initiation ofprotein synthesis.

The term "plasmid" as used herein refers to an extra chromosomal elementoften carrying genes which are not part of the central metabolism of thecell, and usually in the form of circular double-stranded DNA molecules.

The term "restriction endonuclease" refers to an enzyme which binds andcuts within a specific nucleotide sequence within double-stranded DNA.

The term "bioluminescence" refers to the phenomenon of light emissionfrom any living organism.

The term "lux" refers to the lux structural genes which include luxA,luxB, luxC, luxD and luxE and which are responsible for the phenomenonof bacterial bioluminescence. A lux gene complex might include all ofthe independent lux genes, acting in concert, or any subset of the luxstructural genes so long as luxA and luxB are part of the complex.

The term "stress" or "environmental stress" refers to the conditionproduced in a cell as the result of exposure to an environmental insult.

The term "insult" or "environmental insult" refers to any substance orenvironmental change that results in an alteration of normal cellularmetabolism in a bacterial cell or population of cells. Environmentalinsults may include, but are not limited to, chemicals, environmentalpollutants, heavy metals, changes in temperature, changes in pH as wellas agents producing oxidative damage, DNA damage, anaerobiosis, changesin nitrate availability or pathogenesis.

The term "stress response" refers to the cellular response resulting inthe induction of either detectable levels of stress proteins or in astate more tolerant to exposure to another insult or an increased doseof the environmental insult.

The term "stress protein" refers to any protein induced as a result ofenvironmental stress or by the presence of an environmental insult.Typical stress proteins include, but are not limited to those encoded bythe Escherichia coli genes groEL, groES, dnaK, dnaJ, grpE, ion, lysU,rpoD, clpB, clpP, uspA, katG, uvrA, frdA, sodA, sodB, soi-28, narG,recA, xthA, his, lac, phoA, glnA and fabA.

The term "stress gene" refers to any gene whose transcription is inducedas a result of environmental stress or by the presence of anenvironmental insult. Typical E. coli stress genes include, but are notlimited to groEL, groES, dnaK, dnaJ, grpE, lon, lysU, rpoD, clpB, clpP,uspA, katG, uvrA, frdA, sodA, sodB, soi-28, narG, recA, xthA, his, lac,phoA, glnA, micF, and fabA.

The term "heat shock gene" refers to any gene for which its synthesis ispositively controlled by the structural gene encoding the sigma-32protein (rpoH).

The term "stress inducible promoter" refers to any promoter capable ofactivating a stress gene and causing increased expression of the stressgene product.

The term "detector organism" refers to an organism which contains a genefusion consisting of a stress inducible promoter fused to a structuralgene and which is capable of expressing the lux gene products inresponse to an environmental insult. Typical detector organisms includebut are not limited to bacteria.

The term "log phase" or "log phase growth" refers to cell cultures ofdetector organisms growing under conditions permitting the exponentialmultiplication of the detector cell number.

The term "Relative Light Unit" is abbreviated "RLU" and refers to ameasure of light emission as measured by a luminometer, calibratedagainst an internal standard unique to the luminometer being used.

The designation "ATCC" refers to the American Tissue Culture Collectiondepository located in Rockville, Md. The "ATCC No." is the accessionnumber to cultures on deposit at the ATCC.

Environmental insults capable of being detected by the detector organismof the present invention include a variety of organic and inorganicpollutants commonly found in industrial sites, waste streams andagricultural run-off. Such compounds include but are not limited to thepolyaromatic hydrocarbons (PAH), the halogenated aromatics as well as avariety of heavy metals such as lead, cadmium, copper, zinc, and cobalt.Compounds demonstrated to be detected by the method of the presentinvention include atrazine, benzene, copper sulfate,2,4-dichlorophenoxyacetic acid, ethanol, methanol, 2-nitrophenol,4-nitrophenol, pentachlorophenol, phenol, toluene, dimethylsulfoxide,lead nitrate, cadmium chloride, sodium chloride, acetate, propionate,hydrogen peroxide, puromycin, mercury chloride, 2,4-dichloroaniline,propanol, butanol, isopropanol, methylene chloride, Triton X100,acrylamide, methyl viologen, mitomycin C, menadione, ethidium bromide,serine hydroxamate and xylene. Other environmental stresses detectedwere low phosphate levels, poor nitrogen source, poor carbon source andirradiation with ultraviolet light.

The present invention provides a method for the detection ofenvironmental insults at sublethal levels, incorporating a detectororganism containing an expressible gene fusion between a stressinducible promoter and a structural gene resulting in expression of thelux genes.

Detector organisms may include a variety of both prokaryotic andeukaryotic organisms where bacterial cells are preferred.

The present invention provides a stress inducible promoter sensitive tothe presence of an environmental insult. Stress inducible promoters fromboth prokaryotic and eukaryotic cells may be used however promoters frombacteria are preferred and promoters from E. coli are most preferred.Suitable stress inducible promoters may be selected from, but are notlimited to the list of genes under the heading "responding genes" givenin Table I, below:

                  TABLE I                                                         ______________________________________                                                 REGULATORY  REGULATORY  RESPONDING                                   STIMULUS GENE(S)     CIRCUIT     GENES*                                       ______________________________________                                        Protein  rpoH        Heat Shock  grpE, dnaK,                                  Damage.sup.a                     lon, rpoD,                                                                    groESL, lysU,                                                                 htpE, htpG,                                                                   htpI, htpK,                                                                   clpP, clpB,                                                                   htpN, htpO,                                                                   htpX, etc.                                   DNA Damage.sup.b                                                                       lexA, recA  SOS         recA, uvrA,                                                                   lexA, umuDC,                                                                  uvrA, uvrB,                                                                   uvrC, sulA,                                                                   recN, uvrD,                                                                   ruv, dinA,                                                                    dinB, dinD,                                                                   dinF etc.                                    Oxidative                                                                              oxyR        Hydrogen    katG, ahp, etc.                              Damage.sup.c         Peroxide                                                 Oxidative                                                                              soxRS       Superoxide  micF, sodA,                                  Damage.sup.d                     nfo, zwf, soi,                                                                etc.                                         Membrane fadR        Fatty Acid  fabA                                         Damage.sup.e         Starvation                                               Any.sup.f                                                                              ?           Universal   uspA                                                              Stress                                                   Stationary                                                                             rpoS        Resting State                                                                             xthA, katE,                                  Phase.sup.g                      appA, mcc,                                                                    bolA, osmB,                                                                   treA, otsAB,                                                                  cyxAB, glgS,                                                                  dps, csg, etc.                               Amino Acid                                                                             relA, spoT  Stringent   his, ilvBN,                                  Starvation.sup.h                 ilvGMED,                                                                      thrABC, etc.                                 Carbon   cya, crp    Catabolite  lac, mal, gal,                               Starvation.sup.i     Activation  ara, tna, dsd,                                                                hut, etc.                                    Phosphate                                                                              phoB, phoM, P Utilization                                                                             phoA, phoBr,                                 Starvation.sup.j                                                                       phoR, phoU              phoE, phoS,                                                                   aphA, himA,                                                                   pepN, ugpAB,                                                                  psiD, psiE,                                                                   psiF, psiK,                                                                   psiG, psiI,                                                                   psiJ, psiN,                                                                   psiR, psiH,                                                                   phiL, phiO,                                                                   etc.                                         Nitrogen glnB, glnD, N Utilization                                                                             glnA, hut, etc.                              Starvation.sup.k                                                                       glnG, glnL                                                           ______________________________________                                         *Genes whose expression is increased by the corresponding stimulus and        whose expression is controlled by the corresponding regulatory gene(s).       .sup.a Neidhardt and van Bogelen in E. coli and Salmonella typhimurium;       Cellular and Molecular Biology (Neidhardt, F. C., et al. Eds., pp.            1334-1345, American Society of Microbiology, Washington, DC (1987))           .sup.b Walker in E. coli and Salmonella typhimurium; Cellular and             Molecular Biology (Neidhardt, F. C., et al. Eds., pp. 1346-1357, American     Society of Microbiology, Washington, DC (1987))                               .sup.c Christman et al. Cell 41: 753-762 (1985); Storz et al. Science 248     189-194 (1990); Demple, Ann, Rev. Genet. 25: 315-337 (1991)                   .sup.d Demple, Ann. Rev. Genet. 25: 31 337 (1991)                             .sup.e Magnuson et al. Microbiol. Rev 57: 522-542 (1993)                      .sup.f Nystrom and Neidhardt, J. Bacteriol, 175: 2949-2956 (1993); Nystro     and Neidhardt (Mol. Microbiol. 6: 3187-3198 (1992)                            .sup.g Kolter et al. Ann. Rev. Microbiol. 47: 855-874 (1993)                  .sup.h Cashel and Rudd in E. coli and Salmonella typhimurium; Cellular an     Molecular Biology (Neidhardt, F. C., et al. Eds., pp. 1410-1438, American     Society of Microbiology, Washington, DC (1987)); Winkler in E. coli and       Salmonella typhimurium; Cellular and Molecular Biology (Neidhardt, F. C.,     et al. Eds., pp. 395-411, American Society of Microbiology, Washington, D     (1987))                                                                       .sup.i Neidhardt, Ingraham and Schaecter. Physiology of the Bacterial         Cell: A Molecular Approach, Sinauer Associates, Sunderland, MA (1990), pp     351-388; Magasanik and Neidhardt in E. coli and Salmonella typhimurium;       Cellular and Molecular Biology (Neidhardt, F. C., et al. Eds., pp.            1318-1325, American Society of Microbiology, Washington, DC (1987))           .sup.j Wanner in E. coli and Salmonella typhimurium; Cellular and             Molecular Biology (Neidhardt, F. C., et al. Eds., E. coli and Salmonella      typhimurium; Cellular and Molecular Biology (Neidhardt, F. C., et al.         Eds., pp. 1326-1333, American Society of Microbiology, Washington, DC         (1987))                                                                       .sup.k Rietzer and Magasanik in E. coli and Salmonella typhimurium;           Cellular and Molecular Biology (Neidhardt, F. C., et al. Eds., pp.            1302-1320, American Society of Microbiology, Washington, DC (1987));          Neidhardt, Ingraham and Schaecter. Physiology of the Bacterial Cell: A        Molecular Approach, Sinauer Associates, Sunderland, MA (1990), pp 351-388

Table I indicates the relationship of responding gene(s) with aparticular regulatory gene(s) and a regulatory circuit and theassociated cellular stress response triggered by a particular stimulus.

Although the majority of the stress genes listed above in Table I areknown to be positively regulated, the SOS response, produced as a resultof DNA damage, represents a negatively regulated circuit. For definitionof positive and negative control mechanisms see Beckwith in E. coli andSalmonella typhimurium; Cellular and Molecular Biology (Neidhardt, F. C.et al. Eds., pp. 1439-1443, American Society of Microbiology,Washington, D.C. (1987)).

The SOS response to DNA damage is well understood in E. coli. Theproduct of the lexA gene (the LexA repressor) binds to operator elementscontrolling the expression of at least 17 chromosomal genes (Walker inE. coli and Salmonella typhimurium; Cellular and Molecular Biology(Neidhardt, F. C. et al. Eds., pp. 1346-1357, American Society ofMicrobiology, Washington, D.C. (1987)). Upon DNA damage or interferencewith DNA replication, an unknown SOS-inducing signal is produced. Thissignal interacts with the recA gene product converting it into a formthat increases the rate of proteolysis of a limited number of repressormolecules (Little, J. Bacteriol, 175:4943-4950). These repressormolecules are the products of the chromosomal lexA gene and repressorsencoded by and expressed from ultraviolet light-inducible phage genomesin the lysogenic state. SOS promoters are released from repression byRecA protein-mediated proteolysis of the LexA repressor. Among the SOSresponsive promoters are recA and uvrA. It was seen that the recApromoter-lux fusion on a multicopy plasmid produced bioluminescence andresulted in a transformed host cell increasing bioluminescence inresponse to DNA damage. Although the precise mechanism of this result isnot known it is clear that the invention, is not limited to the study ofpositively regulated global regulatory circuits because (1) somenegatively regulated circuits will operate with promoters of respondinggenes in the multicopy state and (2) several means to place negativelycontrolled promoters in a single copy state exist e.g., Oden et al.,Gene 96:29-36 (1990); Symons et al., Gene 53:85-96 (1987); Winans etal., J. Bacteriol, 161:1219-1221 (1985); Arps and Winklet, J. Bacteriol,169:1061-1070 (1987); Jasin and Schimmel, J. Bacterioi, 159:783-786(1984)!.

The invention also provides a transformation vector containing a stressinducible promoter-lux gene fusion, capable of transforming a bacterialhost cell for the expression of the Lux proteins. A variety oftransformation vectors may be used, however, those capable oftransforming E. coli are preferred. pGrpELux.3, pGrpELux.5, pRY001,pRY002, and pRY006 are five specific examples of suitable transformationvectors whose construction is given in detail in the following text.These vectors represent only a sample of the total number of vectorscreated for the purpose of introducing stress promoter-lux reporterfusions into host cells. However, it will be readily apparent to one ofskill in the art of molecular biology that the methods and materialsused in their construction are representative of all other vectorsdescribed. Other preferred vectors are listed in Table V of Example 10.

pGrpELux.3 and pGrpELux.5 are vectors containing the grpE promoter whilepRY001, pRY002 and pRY006 contain the dnaK promoter. pGrpELux.3,pGrpELux.5, and pRY006 were all created by the method of direct cloningwhile PCR protocols were employed as part of the construction method forpRY001 and pRY002. Transformation vectors such as these are common andconstruction of a suitable vector may be accomplished by means wellknown in the art. The preferred source of the lux genes is apre-existing plasmid, containing a promoterless lux gene complex.Similarly, preferred sources of the stress inducible promoter DNA forthe construction of the transformation vector are either also apre-existing plasmid, where the stress inducible promoter DNA is flankedby convenient restriction sites, suitable for isolation by restrictionenzyme digestion, or the product of a PCR reaction.

The pGrpELux.3 and pGrpELux.5, vectors are constructed from the E. colistress gene grpE, and the lux gene complex. pGrpE4 is an E. coli vectorderived from pUC18 (Pharmacia, Cat. No. 27-4949-01). pGrpE4 contains thegrpE gene, including its promoter, bounded at the 5' end by an EcoRIsite and at the 3' end by a BbuI site. Additionally, the grpE promoteris bounded at the 3' end by a PvuII site and an HaeIII site justdownstream of the EcoRI site (FIG. 1). Digestion with EcoRI and BbuIrestriction enzymes yields a 1.1 kb fragment which corresponds to thegrpE gene. Further digestion with PvuII produces two fragments, one ofwhich contains the grpE promoter. The 3' PvuII site on the grpE promoterfragment is converted to an EcoRI site via ligation to phosphorylatedEcoRI linkers. Further digestion by HaeIII yields a grpE promoterfragment conveniently bounded by a 5' HaeIII site and a 3' PvuII site(FIG. 1).

The pUCD615 plasmid containing the lux gene complex is fully describedby Rogowsky et al. (J. Bacteriol, 169 (11) pp 5101-512, (1987)). PlasmidpUCD615 is a 17.6 kb plasmid which contains the genes for kanamycin andampicillin resistance and contains the promoterless lux gene operon(FIG. 1). pUCD615 is first digested with restriction enzymes EcoRI andSmaI, opening the plasmid, followed by ligation with the DNA fragmentsfrom the HaeIII digestion of pgrpE IV.

Typically, the products of the ligation reactions are screened by firsttransforming a suitable host and screening for bioluminescence. Avariety of hosts may be used where hosts having high transformationfrequencies are preferred. XL1Blue (Stratagene, LaJolla, Calif.) andDH5-α (GIBCO-BRL, Gaithersburg, Md.) are two such hosts. Preferredmethods of bioluminescence screening involve exposing gridded culturesof transformants to a suitable X-ray film, followed by visual analysisof the developed films for evidence of exposure. Reisolation of theplasmid from the transformed host and restriction digests followed bygel electrophoresis is used to confirm the existence of the correctplasmid. The plasmids pGrpELux.3 and pGrpELux.5, isolated from twodifferent transformed colonies, are indistinguishable on the basis ofrestriction enzyme analysis. Under some experimental conditions cellstransformed with pGrpELux.5 exhibited higher baseline bioluminescencethan those transformed with pGrpELux.3 and hence pGrpELux.5 is preferredfor the detection of many environmental insults.

The present invention further provides a transformed host cell capableof increased luminescence in the presence of an environmental insult.Many suitable hosts are available where E. coli is preferred and the E.coli strain RFM443 is most preferred. RFM443 is derived from W3102 whichis fully described by B. Bachmann, in E. coli and Salmonellatyphimurium: Cellular and Molecular Biology (Niedhardt et al. Eds., pp1190-1220, American Society of Microbiology, Washington, D.C. (1987)).Transformation of RFM443 by pGrpELux.3 gives the new strain TV1060 whichhas been deposited with the ATCC under the terms of the Budapest Treaty.Transformation of RFM443 by pGrpELux.5 gives the new strain TV1061. Thebaseline of bioluminescence from strain TV1061 is greater than that fromstrain TV1060. E. coli TV1060 has been assigned ATCC No. 69142, andTV1061 has been assigned ATCC No. 69315.

The construction of the plasmid pRY006 containing the dnaK promoterfollowed a similar protocol to that of pGrpELux.3. DNA encoding the dnaKpromoter was obtained from the Lambda phage 9E4 by digestion with therestriction enzymes EcoRI and BamHI. 9E4 is fully described by Kohara etal (Cell 50, 495-508, 1987) herein incorporated by reference.Restriction enzyme digestion produced a 3.7 kb DNA fragment encompassingthe dnaK promoter region bounded on the 5' end by a BamHI site and onthe 3' end by and EcoRI site. As in the construction of pGrpELux.3, thesource of the lux gene complex is pUCD615. pUCD615 was first digestedwith BamHI and EcoRI restriction enzymes followed by ligation with thednaK promoter fragments to produce the plasmid pRY006 (FIG. 2).

Construction of pRY001 and pRY002 is similar to that of pRY006 exceptthat PCR protocols were used to amplify the DNA encoding the dnaKpromoter from 9E4. Briefly, PCR amplification of the dnaK promoter from9E4 was accomplished using the dnaK promoter sequence as described byCowing et al, PNAS 82, 2679-2683, 1985 herein incorporated by reference.

Amplification was carried out as described by the manufacturer (GeneampPCR Reagent Kit, Perkin-Elmer Cetus, Norwalk, Conn.), hereinincorporated by reference. The amplified product corresponding to thednaK promoter region contained convenient BamHI and EcoRI sitesdetermined by the construction of the amplification primers. The dnaKpromoter region was ligated to pUCD615, previously digested withrestriction enzymes BamHI and EcoRI.

Ligated DNA was used to transform E. coli strain DH5α and the resultingtransformants were screened for bioluminescence by exposure to X-rayfilm, and by restriction digests followed by analysis on agarose gels.The strain DH5-α was chosen for this initial screening due to its hightransformation frequency. Two independent colonies were chosen. The twoplasmids isolated from these transformants, pRY001 and pRY002, althoughisolated from independent colonies, are indistinguishable on the basisof restriction enzyme analysis and for the purposes of the presentinvention are considered identical. Under some experimental conditionscells transformed with pRY002 exhibited higher bioluminescence inresponse to environmental insults than those transformed with pRY001 andhence pRY002 is preferred for detection. pRY001, pRY002 and pRY006 werethen used to transform RFM443 to create E. coli stain WM1021, WM1202 andWM1026 respectively. E. coli WM1021, WM1202 and WM1026 have beendeposited with the ATCC under the terms of the Budapest Treaty. E. coliWM1021 has been assigned ATCC No. 69141. E. coli WM1202 has beenassigned ATCC No. 69313. E. coli WM1026 has been assigned ATCC No.69143. As mentioned above, construction of the promoters of other stressgenes fused to the lux reporter was identical to the construction ofpRY001 and pRY002 with the exception that the PCR primers and source oftemplate DNA were different as dictated by the sequences of thepromoters. The sequences of all of the promoters are published and arereadily available through the Genbank database of nucleic acidsequences.

It is well known that hydrophobic compounds are effectively excluded bythe cell envelope from entry into gram negative bacteria, such as E.coli. Recently several E. coli strains containing a mutation fortolerance to colicins (tolC⁻) have been found to have the unexpectedadditional property of increased permeability of host cell envelopes tovarious organic molecules. (Schnaitman et al. J. Bacteriol, 172 (9), pp5511-5513, (1990)). Optionally, it is within the scope of the presentinvention to provide a transformed bacterial host containing the tolC⁻mutation as a suitable detector organism.

In order to create a highly sensitive detector organism with enhancedcell envelope permeability to toxic organics, a tolC⁻ mutation wasintroduced into E. coli strain RFM443.

The E. coli transductant DE112 is isogenic to strain RFM443 except forthe mutation at the tolC locus. It was constructed by phage P1 mediatedgeneralized transduction using a lysate grown on strain CS1562(tolC::miniTn10) (Schnaitman et al. J. Bacteriol, 172 (9), pp 5511-5513,(1990)) as a donor and strain RFM443 as a recipient. Resultanttetracycline resistant transductants were screened for hypersensitivityto the hydrophobic compound crystal violet.

DE112 was transformed with either pGrpELux.5 or pRY002 according tostandard transformation methods as described above to create thedetector organisms TV1076 (grpE lux fusion) and WM1302 (dnaK lux fusion)containing the tolC⁻ mutation. TV1076 and WM1302 have been depositedwith the ATCC under the terms of the Budapest treaty and are designatedATCC No. 69314 and ATCC No. 69316 respectively.

The stress inducible promoter-lux plasmid exists in the transformed hostof the present invention as an autonomously replicating plasmid;however, the routiener will recognize that it is also possible toprovide a transformed host wherein the stress inducible promoter-luxplasmid is integrated into the genome of the transformed host. This maybe accomplished by means known to those skilled in the art. The stressinducible promoter may drive expression of a gene product which in turnactivates expression of the lux gene complex. In this case the promoterand lux gene complex might occur on different genetic elements.

As examples, any of a number of suppression mechanisms may be invokedHartman and Roth, Advances in Genetics, 17:1-105 (1973)!. In one, achromosomally-integrated lux gene complex contains a nonsense mutationin either luxC, luxD, luxA, luxB or luxE and is driven by a constitutivepromoter. The stress inducible promoter is fused such that it drives theexpression of a nonsense suppressor gene. In the absence of stress,bioluminescence is not observed due to organisms' inability tosynthesize the five requisite Lux proteins. If the organism is stressed,the suppressor gene is transcribed. Expression of the suppressor geneproduct allows expression of the five requisite Lux proteins and hencethe organism produces light.

The method of the present invention is designed to allow for themonitoring of samples for the presence of environmental insults by usinga detector organism capable of demonstrating a change in bioluminescencein response to the presence of a insult. The transformed strains TV1060,TV1061, TV1076, WM1021, WM1202, WM1302 and WM1026 are all suitable andpreferred for use as detector organisms. As with the preferred vectors,these detector organisms represent only a sample of the total number ofdetector organisms created for the purpose of detecting environmentalinsults. However, it will be readily apparent to one of skill in the artof molecular biology that the methods and materials used in theirconstruction is representative of all other vectors described. Otherpreferred transformed detector organisms are listed in Table V ofExample 10. At optimum growth conditions a baseline level ofluminescence is produced by the detector organism. Introduction of anenvironmental insult to the actively growing cultures will induce thestress inducible promoter which will in turn activate the lux complex,resulting in an increase in the amount of light emitted from thedetector organism. The amount of light emitted is correlated to thelevel of the insult. For the purpose of the present invention it is mostpreferred if the detector organisms are actively growing in log phasejust prior to exposure to sample suspected of containing an insult andat a cell density of from between about 10 Klett Units to 50 Klett Unitswhere about 20 Klett Units is most preferred. Light emission may bemonitored by a variety of methods capable of detecting photons includingbut not limited to visual analysis, exposure to photographic film,scintillation counting or any device incorporating a photomultipliertube where a luminometer similar to that produced by the DynaTechCorporation is most preferred.

In one embodiment varying concentrations of ethanol were used to applystress to detector organism. As seen in FIG. 4, a final concentration of4% ethanol in the TV1060 cultures (containing the grpE promoter luxfushion) produced a dramatic increase in luminescence at 1000 secondspost-stress. Similarly in FIG. 5, increasing concentrations of ethanolproduced a corresponding increase in luminescence from the stressedcultures. Additionally, the organic pollutants, atrazine,pentachlorophenol (PCP), phenol, 2,4-dichlorophenoxy acetic acid(2,4-D), benzene, methanol, 2-nitrophenol, 4-nitrophenol, atrazine,toluene, dimethylsulfoxide, acetate, propionate, puromycin,2,4-dichloroanaline, propanol, butanol, isopropanol, methylene chloride,Triton X100, acrylamide, methyl viologen, serine hydroxamate, menadione,ethidium bromide, mitomycin C and xylene as well as salts of the heavymetals copper, sulfate, lead nitrate, cadmium chloride, and mercurychloride copper were detected by the present method. Also detected werehigh osmotic strength, ultraviolet (U.V.) light irradiation, theoxidizing agents hydrogen peroxide and growth conditions of limitingphosphate, poor nitrogen source and poor carbon source. Chemicals wereeither dissolved in an appropriate solvent and added to cell culturesfor testing or added directly to the growth media depending on theirsolubility properties. Benzene, ethanol, methanol, propanol,isopropanol, butanol, methylene chloride, dimethyl sulfoxide, TritonX-100, phenol, toluene, and xylene were added directly to LB medium,whereas 2-nitrophenol, 4-nitrophenol, and atrazine were first dissolvedin methanol before being diluted into LB medium. Copper sulfate, leadnitrate, cadmium chloride, mercury chloride, sodium chloride, sodiumacetate, sodium propionate, hydrogen peroxide, puromycin, methylviologen, acrylamide, menadione, ethidium bromide, serine hydroxamate,and mitomycin C were first dissolved in water before being diluted intoLB medium. Pentachlorophenol (PCP), 2,4-dichlorophenoxy acetic acid, and2,4-dichloroaniline were first dissolved in ethanol and finally dilutedinto LB medium for testing. In all cases, the final concentration ofeither ethanol or methanol, or the slight dilution of the medium withwater were such that it did not induce a significant response.

The data shown in FIG. 7 and Tables II and III demonstrate two aspectsof the present detection system. First, that organic and inorganicpollutants can be detected at low concentrations by the instant methodand secondly, that the sensitivity of the system can be enhanced by theuse of a host detector organism containing the tolC⁻ mutation.

FIG. 7 compares the relative sensitivities of detector organismstransformed with the GrpE.Lux.5 fusion with and without the tolC⁻mutation to the presence of pentachlorophenol. As can be seen by thedata, the tolC⁻ mutants exhibit significantly higher sensitivity to thepresence of the PCP than the tolC⁺ parental strain. Table III (Example9) contains data comparing the relative sensitivities of detectororganisms transformed with the pYR002 fusion with and with out the tolC⁻mutation to the presence of PCP, 2,4-D, phenol, atrazine, ethanol,methanol, 2-nitrophenol and copper sulfate. Similarly, it is evidentthat PCP and 2,4-D are preferentially detected by the tolC⁻ mutant host.The tolC⁻ host also appears to be more sensitive to phenol, although toa lesser extent than with PCP and 2,4-D. The tolC⁻ mutation appears tohave little effect on the sensitivity of the detector organism tonon-organic contaminants such as copper sulfate which would be expectedin light of the fact that the tolC⁻ mutation is known to increase thehost cell envelope permeability only to hydrophobic compounds.

Optionally, the method of the present invention may also be used todetect lethal levels of a insult by measuring the decrease in thebaseline luminescence produced by the detector organism. Lethal levelsof insults will interfere with either central metabolism or any luxprotein function of the detector organism, which would be indicated by adecrease in light emitted from the cultures.

FIGS. 6a and 6b illustrate the sensitivity of the transformants WM1021and WM1026 (containing the dnaK promoter lux fushion) to the stress ofvarying concentrations of ethanol. It is interesting to note that at thesublethal concentrations of ethanol varying from 1% to 4%, lightemission increased in a fashion similar to the TV1060 cultures. Bycontrast, lethal concentrations of ethanol in the ranges of 8% to 16%produced a decrease in light emission from the detector cultures.Likewise, higher concentrations of PCP also result in decrease of lightoutput in strain TV1076 (FIG. 7). Thus, it is evident that the method ofthe present invention is capable of a bi-modular function. In one mode,detection of insults at sublethal levels are possible via the mechanismof the induction of the stress inducible promoter and the subsequentincrease in light production from the detector organism. In an alternatemode, levels of insults capable of interfering with central cellularmetabolism can also be detected since bioluminescence is an energy andreducing power dependant phenomenon and any interference with centralmetabolism will cause the baseline luminescence to decrease. Moreover,it is evident that the induction of light production from the stresspromotor-lux fushions occurs at lower concentrations of specificpollutants than those concentrations required to result in a decrease oflight output.

The following non-limiting Examples are meant to illustrate theinvention but are not intended to limit it in any way.

MATERIALS AND METHODS

Restriction enzyme digestions, phosphorylations, ligations andtransformations were done as described in Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Second Edition (1989) Cold Spring HarborLaboratory Press. Isolation of restriction fragments from agarose gelsused Qiagen columns (Qiagen, Inc.) and was performed as specified by themanufacturer. Strataclean (Stratagene, LaJolla, Calif.) and GeneClean(Bio101) were used to remove enzymes from restriction digests, asspecified by the manufacturers. The meaning of abbreviations is asfollows: "h" means hour(s), "min" means minute(s), "sec" meanssecond(s), and "d" means day(s).

EXAMPLES Example 1 Construction of Plasmids pGrpELux.3and pGrpELux.5 andTransformation of RFM443

The outline of the scheme used to construct these plasmids is shown inFIG. 1. FIG. 1 is meant to illustrate the events of the construction,however DNA constructs are not drawn to scale.

Plasmid pGrpE4 was derived from pUC18 (Pharmacia, Cat. No. 27-4949-01)and contains the Escherichia coli stress gene, grpE, including itspromoter sequences. pGrpE4 plasmid was digested with restriction enzymesEcoRI and BbuI and a 1.1 kb fragment was isolated following agarose gelelectrophoresis corresponding to the grpE promoter and structural gene(FIG. 1). The grpE promoter is conveniently bounded on the 5' end by anEcoRI site and on the 3' end by a PvuII site. The isolated fragment wasfurther digested with restriction enzyme PvuII, separating the promoterregion from the structural gene (FIG. 1). An EcoRI linker fragment(Stragene, Catalog #901027) was phosphorylated and then ligated to theproducts of the PvuII digestion replacing the 3' PvuII site on thepromoter with an EcoRI site. Further digestion with HaeIII produced aseries of fragments, one of which contains the grpE promoter bounded onthe 5' end by HaeIII and on the 3' end by EcoRI (FIG. 1).

Plasmid pUCD615 (J. Bacterlol, 169 (11) pp 5101-512, (1987)) is a 17.6kb plasmid which contains the genes for kanamycin and ampicillinresistance and contains the promoterless lux gene operon with multiplecloning sites upstream of the start of lux. (FIG. 1). The pUCD615 isfirst digested with restriction enzymes EcoRI and SmaI, opening theplasmid, followed by ligation with the DNA fragments from the HaeIIIdigestion (FIG. 1).

In order to screen for active grpE-lux fusion ligated DNA was used totransform E. coli strain XL1Blue (Stratagene) by standard CaCl₂transformation protocols and screened for the presence of plasmids usingkanamycin resistance. Colonies were grown in gridded fashion on LBmedium containing kanamycin (25 μg/mL) at 37° C., overnight and werefurther screened for bioluminescence to determine which transformantscontained promoter sequences fused to the lux genes of pUCD615.Bioluminescent screening was done by exposing the gridded colonies toX-OMAT AR film (Kodak, Rochester, N.Y.) at ambient temperature andanalyzing the developed films visually. Confirmation of transformationby the expected plasmid was further obtained by agarose gelelectrophoretic analysis of plasmid DNA from cells producing lightfollowing restriction digests, for the presence and size of restrictionfragments. Restriction fragments from Hind III, BamHI, and Sal Idigestions confirmed the presence and orientation of the grpE promoterin plasmids pGrpELux.3 and pGrpELux.5.

Plasmids pGrpELux.3 and pGrpELux.5. were moved by transformation into E.coli host strain RFM443 to give E. coli strains TV1060 and TV1061respectively. E. coli RFM443 was originally derived from E. coli W3102which is fully described in by B. Bachmann, in E. coli and Salmonellatyphlmurium: Cellular and Molecular Biology (Neidhardt, F. C. et al.Eds., pp 1190-1220, American Society of Microbiology, Washington, D.C.(1987))

Example 2 Construction of Plasmid pRY006 and Transformation of RFM443

The outline of the scheme used to construct pRY006 is shown in FIG. 2.FIG. 2 is meant to illustrate the events of the construction, howeverDNA constructs are not drawn to scale.

DNA containing the dnaK promoter was obtained from Lambda phage 9E4(Kohara, Y. et al; Cell 50, 495-508, 1987) using a Magic Lambda Prep,following the manufacturers suggested protocol (Promega Corp., Madison,Wis.). Phage DNA was digested with restriction enzymes BamHI and EcoRI.Several DNA fragments were liberated by this treatment including a 3.7Kb BamHI-EcoRI restriction fragment encompassing the dnaK promoterregion as described by Cowing et al (PNAS 82, 2679-2683, 1985), andapproximately 3.0 kb of DNA 5' to this region, and 700 bp 3' of thepromoter region encoding the amino terminus of the DnaK protein.Digested DNA was ligated to pUCD615 which had been digested previouslywith restriction enzymes BamHI and EcoRI (FIG. 2). The resulting plasmidconstructs were used to transform E. coli strain DH5-α (GIBCO-BRL,Gaithersburg, Md., Catalog No. 82635a), and transformed bacteria wereplated on LB medium containing 50 μg/mL kanamycin, overnight at 37° C.Resulting colonies were initially screened for bioluminescence byexposure to X-OMAT AR film as described in Example 1. Presence of thedesired plasmid construction was confirmed by restriction enzymeanalysis of plasmid DNA from transformed bacteria. Digestion withrestriction enzymes BamHI and EcoRI yielded a 3.7 Kb restrictionfragment following agarose gel electrophoresis, confirming the presenceof the desired construction. This plasmid was designated pRY006. pRY006was introduced into E. coli strain RFM443 by transformation creatingstrain WM1026.

Example 3 Construction of Plasmid pRY001 and pYR002 and Transformationof RFM443

The outline of the scheme used to construct this plasmid is shown inFIG. 3. FIG. 3 is meant to illustrate the events of the construction;however DNA constructs are not drawn to scale.

DNA containing the dnaK promoter (described by Cowing et al., PNAS 82,2679-2683, 1985) was obtained from Lambda phage 9E4 (Kohara, Y. et al.,Cell 50, 495-508, 1987) using a Magic Lambda Prep (Promega Corp.,Madison, WI), following the protocol as described by the manufacturer.PCR amplification of the dnaK promoter region was accomplished using thefollowing amplification primers:

Upper: 5'-GTTAGCGGATCCAAAAGCACAAAAAAT-3' (SEQ ID NO. 1)

Lower: 5'-AGCAGTGAATTCCATCTAAACGTCTCCA-3' (SEQ ID NO. 2)

DNA amplification was carried out as described by the manufacturer(GeneAmp PCR Reagent Kit, Perkin-Elmer Cetus, Norwalk, Conn.). Reagentconcentrations were:

1×Buffer

200 uM dATP

200 uM dCTP

200 uM dGTP

200 uM dTTP

2.5 units Amplitaq Polymerase (Perkin-Elmer Cetus)

100 pM Upper Primer

100 pM Lower Primer

1 ng 9E4 phage DNA Template

dH₂ O to 100 μL

The reaction was performed using a Perkin-Elmer Cetus GeneAmp PCR System9600 thermal cycler programmed as follows:

Melting: 94° C. for 10 sec

Annealing: 50° C. for 10 sec

Extension: 72° C. for 15 sec

Cycles: 30

The amplified product which results is 207 base pairs in length, andcontains the entire 182 bp segment encoding the dnaK promoter region asdeposited in GeneBank (Accession 10420; Locus ECODNAK), as well as short5' and 3' flanking sequences. PCR-amplified DNA was digested withrestriction enzymes BamHI and EcoRI, and ligated to pUCD615 previouslydigested with restriction enzymes BamHI and EcoRI (FIG. 3). Theresulting plasmid constructs were introduced into the E. coli strainDH5-α (GIBCO-BRL, Gaithersburg, Md., Cat. No. 82635a) by standardtransformation protocols, and bacteria were plated on LB mediumcontaining 50 μg/mL kanamycin and grown overnight at 37° C. Resultingcolonies were initially screened for bioluminescence by exposure toX-ray film as described in Example 1. Two colonies were picked fortransformation analysis and the presence of the desired plasmidconstruction was confirmed by restriction enzyme analysis of plasmid DNAfrom transformed bacteria. Appearance of a 193 bp BamHI-EcoRIrestriction fragment following agarose gel electrophoresis confirmed thepresence of the desired construction. These plasmids were designatedpRY001 and pRY002. Although pRY001 and pRY002 represent differenttransformation colonies they are indistinguishable on the basis ofrestriction enzyme mapping and for the purposes of the present inventionare considered identical. pRY001 and pRY002 were introduced into E. colistrain RFM443 by transformation creating strains WM1021 and WM1202,respectively.

Example 4 Stress Induction of Bioluminescence at 4% Ethanol

Strain TV1060 was grown at 37° C. in LB medium containing kanamycin (25μg/mL) until it reached Klett 56 (measured on a Klett-Summersoncolorimeter with a #66 red filter) at which time it was diluted 1:11into the same medium at ambient temperature and allowed to grow for 3 hat ambient temperature until reaching a density of 20 Klett Units. 100μL of cells were placed into the wells of a microtiter plate followed bythe addition of either 10 μL 40% ethanol (experimental, finalconcentration 4% ethanol) or 10 μL of distilled water (control). Theplate was immediately placed into a luminometer (Luminoskan, Finland)and bioluminescence was measured as RLU vs. time. As can be seen in FIG.4, light emission from the transformed TV1060 cultures stressed with 4%ethanol increased dramatically 1000 sec post-stress reaching a maximumlevel of 130 RLU. Addition of the distilled water to the controlcultures produced no variation in the baseline luminescence (FIG. 4).

Example 5 Stress Induction of Bioluminescence at Varying Concreations ofEthanol

Strain TV1060 was grown overnight in LB medium containing kanamycin (25μg/mL) and then diluted 1:100 in the same medium and grown at roomtemperature until reaching a Klett of 20. 100 μL of cells were placedinto the wells of a microtiter plate containing 100 μL of either 2%, 4%,8% or 16% (giving final concentrations of 1%, 2%, 4%, and 8%respectively of ethanol, experimental) or 100 gL of the same medium(control). The plate was immediately placed into a luminometer, modelML3000 (Dynatech Laboratories, Chantilly, Va.) and bioluminescence wasmeasured as RLU vs. time. As can be seen in FIG. 5, light emission fromthe transformed TV1060 cultures stressed with ethanol demonstrated anincrease in luminescence corresponding to increasing concentrations ofethanol. As in Example 4, increases in luminescence were observed after1000 sec post-stress. Control cultures produced no variation in thebaseline luminescence (FIG. 5). Maximum light emission was obtained with8% ethanol at 3000 sec post-stress showing a 263 fold increase in lightproduction over the control. Lower levels of ethanol stress producedcorrespondingly lower levels of light output where 4%, 2%, and 1%ethanol concentrations gave 96, 13.5 and 3.9 fold increases in lightemission over the controls, respectively.

Example 6 Stress Induction of Bioluminescence in Strains WM1021 andWM1026

Strains WM1021 and WM1026 were grown at 37° C. for approximately 18 h inLB medium containing kanamycin (50 μg/mL). Cultures were then diluted1:50 into the fresh media, and grown at ambient temperature forapproximately 3 h. When cells reached a density of Klett Units of 20, 80μL of the cell suspension was placed into the wells of a microtiterplate containing 20 μL of ethanol at various concentrations, and theplate was immediately placed into a Dynatech Model ML3000 luminometer.Ethanol was present at final concentrations of 16%, 8%, 4%, 2%, and 1%,and a deionized water control was included. The luminometer waspreviously programed to measure luminescence at 2 min intervals. FIGS.6a and 6b present the resulting luminescence data produced in responseto varying concentrations of ethanol by strains WM1021 and WM1026,respectively. As shown in FIG. 6a luminescence increases sharply 1000sec post-stress in those cultures receiving the sublethal concentrationsof 1%, 2% and 4% ethanol. At the higher, lethal concentrations ofethanol of 8% and 16%, luminescence was seen to decline below baselinevalues, suggesting cell death. FIG. 6b shows similar results using thehost cell WM1026. Thus, light is increased in response to sublethallevels of ethanol whereas light output is diminished at higher, lethallevels. This example demonstrates the ability of the invention to detectthe presence of both sublethal and lethal levels of an insult.

Example 7 Comparison of Stress Induction of Bioluminescence byPentachlorophenol in tolC⁺ and tolC⁻ Strains Transformed withpGrpE.Lux.5

In order to provide an E. coli detector organism with enhancedpermeability to organic compounds the tolC⁻ mutant DE112 was constructedfrom RFM443. The E. coli strain DE112 is isogenic to strain RFM443except for the mutation at the tolC locus. It was constructed by phageP1 mediated transduction using a lysate grown on strain CS1562(tolC::miniTn10) as a donor and strain RFM443 as a recipient. CS1562(tolC::miniTnlO) is fully described in Austin et al., J. Bacteriol. 172,5312, (1990) and the transduction procedure is described in Miller, J.H., Experiments in Molecular Genetics, (1972) Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., pp 201-205. RFM443 was originallyderived from W3102 which is fully described in by B. Bachmann, in E.coli and Salmonella typhimurium: Cellular and Molecular Biology(Niedhardt et al. Eds., pp 1190-1220, American Society of Microbiology,Washington D.C., (1987)). Resultant tetracycline resistant recombinantswere screened for hypersensitivity to the hydrophobic compound crystalviolet.

DE112 and RFM443 are isogenic except at the tolC locus. Both strainswere transformed with the plasmid pGrpELux.5 as described in Example 1,and designated TV1076 (tolC::miniTn10) and TV1061 (tolC⁺) respectively.

Both strains were grown overnight at 25° C. in LB medium containingKanamycin at 50 μg/mL. The overnight cultures were diluted 1:50 intofresh LB medium containing Kanamycin (50 μg/mL) and were incubated withshaking at 25° C. for 4 h. Cell density was determined on aKlett-Summerson colorimeter. When both strains reached readings ofbetween 25 and 29 Klett Units the cells were used for testing.

Cells (50 μL) from each culture were added to LB medium containingKanamycin (50 μg/mL) and various concentrations of pentachlorophenol(PCP) in the wells of a microtiter plate such that the final volume ineach well was 100 μL.

A 100 mg/mL stock solution of PCP in ethanol was used to make a 75 μg/mLsolution of PCP in LB medium containing Kanamycin (50 μg/mL). Thissolution (50 μL) was placed in wells of a microtiter plate and serialtwo-fold dilutions were made of from these. When the equal volume ofcells was added, the highest ethanol concentration was 0.037%, aconcentration known not to induce a significant response from thesecells.

Light readings were taken at periodic intervals, in a Dynatech ML3000microtiter plate luminometer at 25° C., and the data is shown in thegraph of FIG. 7 plotted as concentration of PCP as a function ofinduction ratio. The induction ratio is defined as the light output(RLU) in the presence of PCP divided by the light output (RLU) in theabsence of PCP.

FIG. 7 shows the induction ratio at 60 min after the addition vs. thedose of PCP. At low doses, no induction of light output (ratio=1) fromthe tolC⁺ strain, TV1061, was seen. However, at these lower doses thetolC⁻ strain, TV1076, showed significant increase of light output. Also,at higher doses, the induction in the tolC⁺ strain is observed, and atoxic effect of the PCP (loss of light output) is observed in the tolC⁻strain. Thus, the tolC⁻ strain is useful for detecting lowerconcentration of this compound than can be detected in the tolC⁺ strain.Likely, other hydrophobic compounds will also be more readily detectedby the combination of this host strain with the plasmids containinggrpE:: lux fusions

Example 8 Stress Induction of Bioluminescence by Organic and InorganicPollutants in tolC⁺ and tolC⁻ Strains Transformed with PGrpE.Lux.5

TV1061 and TV1076 were engineered as described above. Both strains weregrown overnight at 25° C. in LB medium containing Kanamycin at 50 μg/mL.The cover night cultures were diluted between 40 and 100-fold into freshLB medium containing Kanamycin (50 μg/mL) and were incubated withshaking at 25° C. Cell density was determined on a Klett-Summersoncolorimeter. When both strains gave readings of between 15 and 30 KlettUnits, cells were used for testing.

Cells (50 BL) from each culture were added to LB medium containingKanamycin (50 μg/mL) and various concentrations of benzene, ethanol,methanol, 2-nitrophenol, 4-nitrophenol, PCP, phenol, toluene, and xylenein the wells of a microtiter plate such that the final volume in eachwell was 100 BL. Control wells contained appropriate volumes of solventsused to dissolve the test compounds.

Benzene, ethanol, methanol, propanol, isopropanol, butanol, methylenechloride, dimethyl sulfoxide, Triton X-100, phenol, toluene, and xylenewere added directly to LB medium containing kanamycin (50 μg/mL). Stocksolutions in methanol of 2-nitrophenol (136 mg/mL) and of 4-nitrophenol(112 mg/mL) were diluted into LB medium containing kanamycin (50 μg/mL)to concentrations tested. The final concentration of methanol was suchthat it did not induce a significant response form these cells. A stocksolution in ethanol of PCP (100 mg/mL) was diluted into LB mediumcontaining kanamycin (50 Bg/mL) to the concentration tested. The finalconcentration of ethanol was such that it did not induce a significantresponse from these cells. Stock solutions in water of copper sulfate(250 mM), lead nitrate (100 mM), mercury chloride (100 mM), sodiumchloride (20%), sodium acetate (2M), sodium propionate (2M), hydrogenperoxide (30%), puromycin (10 mg/mL), methyl viologen (200 mg/mL), andacrylamide (1M) were diluted into LB medium containing kanamycin (50μg/mL) to the concentrations tested. A stock solution in water ofcadmium chloride (100 mM) was diluted into LB medium lacking kanamycinto the concentrations tested.

Light readings were taken at periodic intervals over a two h period andthe data is shown in Table II. All luminescence readings were measuredin a Dynatech ML3000 microtiter plate luminometer at 25° C.

Data in Table II represent readings taken 1 h post exposure to the testcompounds and are expressed as the concentration of test compound thatgives the maximum luminescence. Data are also given showing the foldincrease in induced luminescence over the baseline luminescence.

                                      TABLE II                                    __________________________________________________________________________    Induction of Light Production from Plasmid pGrpELux.5 by Chemicals                          TV1061 (tolC.sup.+)                                                                         TV1076 (tolC.sup.-)                                             Conc. for Fold                                                                              Conc. for Fold                                    Chemical      Max Induction                                                                           Increase                                                                          Max Induction                                                                           Increase                                __________________________________________________________________________    Benzene       10   mg/mL*                                                                             1.8 10   mg/mL*                                                                             2                                       Ethanol       2%   (v/v)                                                                              182 2%   (v/v)                                                                              140                                     Methanol      4%   (v/v)                                                                              40  4%   (v/v)                                                                              17                                      2-nitrophenol 113  μg/mL                                                                           2.5 113  μg/mL                                                                           2.5                                     4-nitrophenol 37   μg/mL                                                                           4.7 37   μg/mL                                                                           3                                       Pentachlorophenol                                                                           37.5 μg/mL*                                                                          15.3                                                                              1.17 μg/mL                                                                           6.7                                     Phenol        460  μg/mL                                                                           12  460  μg/mL                                                                           11.1                                    Toluene       10   mg/mL*                                                                             2.7 10   mg/mL*                                                                             3.2                                     Xylene        3.3  mg/mL                                                                              1.7 3.3  mg/mL                                                                              1.4                                     2,4-Dichlorophenoxy acetic acid                                                             133  μg/mL                                                                           13.1                                                                              133  μg/mL                                                                           9.3                                     Copper Sulfate                                                                              1000 μg/mL                                                                           6.3 1000 μg/mL                                                                           5.5                                     Dimethyl Sulfoxide                                                                          1%   (v/v)*                                                                             2.9 1%   (v/v)                                                                              2.6                                     Lead Nitrate  800  μM*                                                                             2.4                                                   Sodium Chloride                                                                             2%   (w/v)*                                                                             9.2                                                   Cadmium Chloride                                                                            333  μM                                                                              9.7                                                   Sodium Acetate                                                                              50   mM   14                                                    Sodium Propionate                                                                           100  mM*  76                                                    Hydrogen Peroxide                                                                           0.0093%                                                                            (w/v)                                                                              54.5                                                                              0.0046%                                                                            (w/v)                                                                              6.1                                     Puromycin     50   μg/mL                                                                           1.6 Only inhibition                                                               detected                                          Mercury Chloride                                                                            0.39 μM                                                                              1.4 0.39 μM                                                                              1.6                                     2,4-dichloroaniline                                                                         100  μg/mL*                                                                          49.4                                                                              100  μg/mL*                                                                          49.4                                    Propanol      2%   (v/v)                                                                              84.8                                                  Isopropanol   2%   (v/v)                                                                              124                                                   Butanol       0.5% (v/v)                                                                              27.7                                                  Methylene Chloride                                                                          4%   (v/v)                                                                              11.4                                                  Triton X-100  1%   (v/v)*                                                                             1.2                                                   Methyl Viologen                                                                             50   μg/mL                                                                           2                                                     Acrylamide    50   mM*  1.9                                                   __________________________________________________________________________     *Maximum concentration tested                                            

It is evident from the data in Table II that a wide variety of chemicalcompounds and environmental conditions will induce bioluminescence fromE. coli strains containing the plasmid pGrpELux.5. Hence, it may beconcluded that detector organisms containing the tolC⁻ mutationrepresent a preferred host for some environmental insults.

Example 9 Stress Induction of Bioluminescence by Organic and InorganicPollutants in tolC⁺ and tolC⁻ Strains Transformed with pRY002

DE112 (tolC::miniTn10) was engineerd as described in Example 7, and thesource of RFM443 has beeen previously discussed. Both strains weretransformed with the plasmid pRY002 by standard methods as described inExample 3, and designated WM1302 (tolC::miniTn10) and WM1202 (tolC⁺)respectively.

Both strains were grown overnight at 25° C. in LB medium containingKanamycin at 50 μg/mL. The overnight cultures were diluted 1:100 intofresh LB medium containing Kanamycin (50 μg/mL) and were incubated withshaking at 25° C. for 4 h. Cell density was determined on aKiett-Summerson colorimeter. When both strains gave readings of 28 KlettUnits, cells were used for testing.

Cells (50 μL) from each culture were added to wells of a microtiterplate containing LB medium containing Kanamycin (50 μg/mL) and variousconcentrations of PCP, 2,4-dichlorophenoxyacetic acid (2,4-D), phenol,ethanol, methanol, 2-nitrophenol, atrazine and copper sulfate such thatthe final volume in each well was 100 μL. Control wells containedappropriate volumes of solvents used to dissolve the test compounds.

Chemicals were prepared for testing essentially as described in Example8. Copper sulfate was added directly to the culture medium and atrazinewas first dissolved in methanol before additions to cell cultures. Withthe exception of atrazine, all solvents were added at levels below thoseneeded to see a stress response in the system. The level of methanolpresent in the atrazine sample induced a low but detectable response;data in Table III is the net response detected above the responseobtained with an equivalent amount of methanol alone.

Light readings were taken at periodic intervals over a two h period andthe data is shown in Table III. All luminescence readings were measuredin a Dynatech ML3000 microtiter plate luminometer at 25° C.

Data in Table III represent readings taken 1 h post exposure to the testcompounds and are expressed as the concentration of test compound thatgives the maximum luminescence. Data are also given showing the foldincrease in induced luminescence over the baseline bioluminescence.

                  TABLE III                                                       ______________________________________                                        Induction of Bioluminescence from Cells                                       Transformed with Plasmid pRY002 by Chemicals                                         WM1202 (tolC.sup.+)                                                                         WM1302 (tolC.sup.-)                                               Conc. for   Fold    Conc. for Fold                                   Chemical Max Ind.**  Incr.***                                                                              Max. Ind. Incr.                                  ______________________________________                                        Atrazine 135     μg/mL*                                                                             2.2   no ind.   --                                   Copper Sulfate                                                                         3.3     mM*     9     3.3   mM*   8.4                                Ethanol  4%      (v/v)   1264  4%    (v/v) 1525                               2,4-D    400     μg/mL*                                                                             42    100   μg/mL                                                                            140                                Methanol 5%      (v/v)   3.7   5%    (v/v) 3.9                                2-nitrophenol                                                                          340     μg/mL*                                                                             7.6   340   μg/mL*                                                                           9.3                                PCP      37.5    μg/mL*                                                                             106   1.2   μg/mL                                                                            45                                 Phenol   1.4     mg/mL   6     1.4   mg/mL 72                                 ______________________________________                                         *Maximum concentration tested                                                 **"Conc. for Max. Ind." means concentration for maximum induction             ***"Fold Incr." means the fold increase over baseline bioluminescence    

It is evident from the data in Table III that several classes ofchemical compounds will induce bioluminescence from E. coli strainscontaining the plasmid pRY002. Both the tolC⁻ and tolC+ hostsdemonstrated similar sensitivities to less hydrophobic compounds. It maybe concluded that detector organisms containing the tolC⁻ mutationrepresent a preferred host for some environmental insults.

Example 10 Construction of Promoters lon, recA, uvrA, katG, micF, uspA,xthA, his, lac, phoA, glnA Fused to the lux operon

The scheme to construct additional plasmids is identical to theconstruction of pRY001 and pRY002 as illustrated in FIG. 3 and describedin Example 3, with the exception that different primers and templateswere used for the PCR reactions, and that 40 cycles were used in PCRreactions. The primers and templates used are listed below in Table IV.

                                      TABLE IV                                    __________________________________________________________________________                                                               Product            Target                                                                             Upper Primer                Lower Primer           Tem-                                                                             Size               Promoter.sup.a                                                                     (5' to 3').sup.b            (5' to 3').sup.c       plate.sup.d                                                                      (bp).sup.c         __________________________________________________________________________    lon  ACTTAAGGATCCAAGCGATGGCGCGTAAAA SEQ ID NO:3                                                                AGCAGCGAATTCATCGCCGCTTCCAGACAA                                                                       148                                                                              534                                                 SEQ ID NO:4                                  recA ACTTAAGGATCCAGAGAAGCCTGTCGGCAC SEQ ID NO:5                                                                AGCTTTGAATTCCGCTTCTGTTTGTTTT                                                                         C  279                                                 SEQ ID NO:6                                  uvrA ACTTTTGGATCCGTGTAAACGCGCGATTG SEQ ID NO:7                                                                 AGCAGCGAATTCTTCCCGGATTAAACGCTT                                                                        637,                                                                            225                                                 SEQ ID NO:8            638                   katG ACTTAAGGATCCCGAAATGAGGGCGGGAAA SEQ ID NO:9                                                                AGCAGCGAATTCGAACGTTGCTGACCACGA                                                                       538                                                                              654                                                 SEQ ID NO:10                                 micF ACTTAAGGATCCCCCCAAAAATGCAGAATA SEQ ID NO:11                                                               AGCAGCGAATTCGGGCATCCGGTTGAAATAG                                                                      373                                                                              331                                                 SEQ ID NO:12                                 fabA ACTTAAGGATCCGCCATTACGTTGGCTGAA SEQ ID NO:13                                                               AGCAGCGAATTCCCACCCGTTTCGGTCATT                                                                       222                                                                              260                                                 SEQ ID NO:14                                 uspA ACTTAAGGATCCCTCCCGATACGCTGCCA SEQ ID NO:15                                                                AGCAGCGAATTCGGCGATGAGAATGTGTTTAT                                                                     608                                                                              381                                                 SEQ ID NO:16                                 xthA ACTTAAGGATCCAATTACTGCGCCATTCTG SEQ ID NO:17                                                               ACATCGGAATTCTCATAGTCGCTGCCATTT                                                                       C  181                                                 SEQ ID NO:18                                 his  ACTTAAGGATCCCTAATTGTACGCATGTCA SEQ ID NO:19                                                               AGCAGCGAATTCAAAGTCTCTGTGAATGTT                                                                       350                                                                              301                                                 SEQ ID NO:20                                 phoA ACTTAAGGATCCAGATTATCGTCACTGCAA SEQ ID NO:21                                                               AGCAGCGAATTCGGCCAATCAGCAAAATAA                                                                        141,                                                                            501                                                 SEQ ID NO:22           142                   glnA ACTTTCGGATCCTTGGTGCAACATTCACAT SEQ ID NO:23                                                               AGCAGCGAATTCTCAGCGGACATCGTCAGT                                                                       546                                                                              227                                                 SEQ ID NO:24                                 __________________________________________________________________________     .sup.a Promoter sequences are available in Genbank.                           .sup.b The underlined region of the upper primer is BamHI restriction         enzyme cleavage site. 3' to the restriction site is sequence (generally 1     nucleotides) complimentary the region upstream of the desired promoter.       .sup.c The underlined region of the lower primer is an EcoRI restriction      enzyme cleavage site. 3' to the restriction site is sequence (generally 1     nucleotides) complimentary to the region downstream of the desired            promoter.                                                                     .sup.d The template for PCR reactions was either a chromosomal DNA            preparation (C, prepared according Zyskind & Bernstein, Recombinant DNA       Laboratry Manual, Academic Press, New York, 1992) or a waterresuspended       plaque from one of an overlapping set of specialized λ transducing     phages coveringthe E. coli chromosome  Kohara et al. Cell 50; 495-508         (1987)! indicated by its assigned number  see Bouffard et al., CABIOS 8:      563-567 (1992)! growth on E. coli K12.Amplification from plaques was          performed by the method of Berg  Krishnan, Blakesley and Berg (1992)          Nucleic Acids Reaseacrh 19: 1153! while that from the chromosmal              preparation differed from that of Berg that 1 μl of the chromosomal DN     preparation was used as template.                                             .sup.e The calculated size of the PCR product. Agarose gel electrophoreti     analysis confirmed product size.                                         

All of the promoter sequences listed in Table IV are published andavailable from GenBank. The underlined region of the upper primer is aBamHI restriction enzyme cleavage site. 3' to the restriction site issequence (generally 18 nucleotides) complimentary to the region upstreamof the desired promoter. The underlined region of the lower primer is anEcoRI restriction enzyme cleavage site. 3' to the restriction site issequence (generally 18 nucleotides) complementary to the regiondownstream of the desired promoter. The template for PCR reactions waseither a chromosomal DNA preparation (C, prepared according to Zyskind &Bernstein, Recombinant DNA Laboratory Manual, Academic Press, New York,1992) or a water resuspended plaque from one of an overlapping set ofspecialized λ transducing phages covering the E. coli chromosome (Koharaet al., Cell 50; 495-508 (1987)) indicated by its assigned number(Bouffard et al., CABIOS 8: 563-567 (1992)) grown on E. coli K12.Amplification from plaques was performed by the method of Berg et al.,Nucleic Acids Research 19,1153, (1991) Amplifications from thechromosomal preparations differed from that of Berg in that 1 μL of thechromosomal DNA preparation was used as template. The calculated size ofthe PCR product was confirmed by agarose gel electrophoresis.

Each of the resultant plasmids were placed in three E. coli hoststrains: RFM443, DE112, and W3110. Plasmids and transformed host cellsare listed below in Table V.

                  TABLE V                                                         ______________________________________                                        Promoter  Plasmids   E. coli host                                                                             Strain name                                   ______________________________________                                        lon       pLonE6     W3110      pLonE6/W3110                                            pLonE6     RFM443     pLonE6/RFM443                                           pLonE6     DE112      pLonE6/DE112                                            pLonF1     W3110      pLonF1/W3110                                            pLonF1     RFM443     pLonF1/RFM443                                           pLonF1     DE112      pLonF1/DE112                                  recA      pRecALux1  W3110      DPD2789                                                 pRecALux2  W3110      DPD2790                                                 pRecALux3  W3110      DPD2791                                                 pRecALux1  RFM443     DPD2792                                                 pRecALux2  RFM443     DPD2793                                                 pRecALux3  RFM443     DPD2794                                                 pRecALux1  DE112      DPD2795                                                 pRecALux2  DE112      DPD2796                                                 pRecALux3  DE112      DPD2797                                       uvrA      pUvrALux1  W3110      DPD2814                                                 pUvrALux2  w3110      DPD2815                                                 pUvrALux3  W3110      DPD2816                                                 pUvrALux4  W3110      DPD2817                                                 pUvrALux1  RFM443     DPD2818                                                 pUvrALux2  RFM443     DPD2819                                                 pUvrALux3  RFM443     DPD2820                                                 pUvrALux4  RFM443     DPD2821                                                 pUvrALux1  DE112      DPD2622                                                 pUvrALux2  DE112      DPD2823                                                 pUvrALux3  DE112      DPD2824                                                 pUvrALux4  DE112      DPD2825                                       katG      pKatGLux2  W3110      DPD2507                                                 pKatGLux6  W3110      DPD2508                                                 pKatGLux2  DE112      DPD2509                                                 pKatGLux6  DE112      DPD2510                                                 pKatGLux2  RFM443     DPD2511                                                 pKatGLux6  RFM443     DPD2512                                       micF      pMicFLux1  W3110      DPD2515                                                 pMicFLux2  W3110      DPD2516                                                 pMicFLux1  DE112      DPD2517                                                 pMicFLux2  DE112      DPD2518                                                 pMicFLux1  RFM443     DPD2519                                                 pMicFLux2  RFM443     DPD2520                                       uspA      pUspALux.2 W3110      DE130                                                   pUspALux.2 RFM443     DE134                                                   pUspALux.2 DE112      DE138                                                   pUspALux.6 W3110      DE142                                                   pUspALux.6 RFM443     DE146                                                   pUspALux.6 DE112      DE150                                                   pUspALux.13                                                                              W3110      DE154                                                   pUspALux.13                                                                              RFM443     DE158                                                   pUspALux.13                                                                              DE112      DE162                                         xthA      pXthALux1  W3110      DPD2771                                                 pXthALux2  W3110      DPD2772                                                 pXthALux3  W3110      DPD2773                                                 pXthALux4  W3110      DPD2774                                                 pxthALux5  W3110      DPD2775                                                 pXthALux6  W3110      DPD2776                                                 pXthALux1  RFM443     DPD2777                                                 pXthALux2  RFM443     DPD2778                                                 pXthALux3  RFM443     DPD2779                                                 pXthALux4  RFM443     DPD2780                                                 pXthALux5  RFM443     DPD2781                                                 pXthALux6  RFM443     DPD2782                                                 pXthALux1  DE112      DPD2783                                                 pXthALux2  DE112      DPD2784                                                 pXthALux3  DE112      DPD2785                                                 pXthALux4  DE112      DPD2786                                                 pXthALux5  DE112      DPD2787                                                 pXthALux6  DE112      DPD2788                                       his       pHisLux5   RFM443     DPD1534                                                 pHisLux9   RFM443     DPD1535                                                 pHisLux12  RFM443     DPDI536                                                 pHisLuxS   W3110      DPD1537                                                 pHiSLux9   W3110      DPD1538                                                 pHiSLux12  W3110      DPD1539                                                 pHiSLux5   DE112      DPD1540                                                 pHiSLux9   DE112      DPD1541                                                 pHisLux12  DE112      DPD1542                                       lac       pLacLux    W3110      TV1063                                                  pLacLux    RFM443     TV1058                                                  pLacLux    RFM443     TV1068                                                  pLacLux    DE112      TV1073                                        phoA      pPhoALux3  W3110      DPD1522                                                 pPhoALux4  W3110      DPD1523                                                 pPhoALux5  W3110      DPD1524                                                 pPhoALux11 W3110      DPD1525                                                 pPhoALux3  RFM443     DPD1526                                                 pPhoALux4  RFM443     DPD1527                                                 pPhoALux5  RFM443     DPD1528                                                 pPhoALux11 RFM443     DPD1529                                                 pPhoALux3  DE112      DPD1530                                                 pPhoALux4  DE112      DPD1531                                                 pPhoALux5  DE112      DPD1532                                                 pPhoALux11 DE112      DPD1533                                       glnA      pGlnALux1  W3110      DPD2830                                                 pGlnALux2  W3110      DPD2831                                                 pGlnALux3  W3110      DPD2832                                                 pGlnALux4  W3110      DPD2833                                                 pGlnALux1  RFM443     DPD2834                                                 pGlnALux2  RFM443     DPD2835                                                 pGlnALux3  RFM443     DPD2836                                                 pGlnALux4  RFM443     DPD2837                                                 pGlnALux1  DE112      DPD2838                                                 pGlnALux2  DE112      DPD2839                                                 pGlnALux3  DE112      DPD2840                                                 pGlnALux4  DE112      DPD2841                                       ______________________________________                                    

Promoter-reporter fusions were tested in transformed detector host cellsusing a variety of environmental insults, appropriate to the knownsensitivity of the promoter. Promoters and their corresponding inducinginsults are summarized in Table VI.

                  TABLE VI                                                        ______________________________________                                        Target             Observed       Expression                                  Promoter                                                                              Upset.sup.b                                                                              Induction by.sup.c                                                                           lacking in.sup.d                            ______________________________________                                        lon     protein damage                                                                           ethanol.sup.e, copper sulfate                              recA    DNA damage mitomycin C.sup.f, cadmium                                                    chloride.sup.g, U.V. light                                                    ethidium bromide                                           uvrA    DNA damage U.V. light                                                 katG    oxidative  methyl viologen.sup.h,                                             damage     hydrogen peroxide.sup.i,                                                      menadione.sup.h                                            micF    oxidative  methyl viologen,                                                   damage     hydrogen peroxide                                          uspA    any        ethanol.sup.j, copper sulfate                              xthA    stationary acetate, propionate,                                               phase      hydrogen peroxide                                          his     amino acid serine hydroxamate.sup.k                                                                     relA spoT                                           starvation                                                            lac     carbon     absence of glucose as a                                            starvation C source.sup.1                                             phoA    phosphate  low phosphate levels.sup.m                                         limitation                                                            glnA    nitrogen   glutamine as sole N                                                limitation source.sup.n                                               ______________________________________                                         .sup.b causing a pleiotropic regulatory response.                             .sup.c chemical induction turning on an increased bioluminescent response     .sup.d genetic construction preventing bioluminescent expression by           disruption of a positive regulatory circuit.                                  .sup.e ethanol is a strong inducer of the heat shock response  Niedhardt      and VanBogelen in E. coli and Salmonella typhimurium; Cellular and            Molecular Biology (Neidhardt, F. C. et al. Eds., pp. 1334-1345, American      Society of Microbiology, Washington, DC (1987)!                               .sup.f mitomycin C is a known inducer of the SOS response  Walker in E.       coli and Salmonella typhimurium; Cellular and Molecular Biology               (Neidhardt, F. C. et al. Eds., pp. 1346-1357, American Society of             Microbiology, Washington, DC (1987)!.                                         .sup.g cadmium had been reported to induce genetic damage  Neidhardt and      VanBogelen in E. coli and Salmonella typhimurium; Cellular and Molecular      Biology (Neidhardt, F. C. et al. Eds., pp. 1334-1345, American Society of     Microbiology, Washington, DC (1987)!.                                         .sup.h this compound promotes redox cycling producing superoxide.             Superoxide is dismutated to hydrogen peroxide. Superoxide induces             synthesis of 40 proteins in addition to the 40 proteins induced by            exposure to hydrogen peroxide  Demple, Ann. Rev. Genet. 25: 315-337           (1991)!.                                                                      .sup.i hydrogen peroxide induces the oxyR regulon and several other           proteins among the approximately 40 polypeptides induced in E. coli and S     typhimurium  Christman et al. Cell 41: 753-762 (1985); Storz et al.           Science 248: 189-194 (1990); Demple, Ann. Rev. Genet. 25: 315-337 (1991)!     .sup.j T. Van Dyk, unpublished.                                               .sup.k this chemical prevents aminoacylation of tRNA.sup.ser thus inducin     the stringest response  Cashel and Rudd in E. coli and Salmonella             typhimurium; Cellular and Molecular Biology (Neidhardt, F. C. et al. Eds.     pp. 1410-1438, American Society of Microbiology, Washington, DC (1987);       Winkler in E. coli and Salmonella typhimurium; Cellular and Molecular         Biology (Neidhardt, F. C. et al. Eds., pp. 395-411, American Society of       Microbiology, Washington, DC (1987)!.                                         .sup.l the catabolite activation response is triggered by the absence of      good carbon source  Neidhardt, Ingraham and Schaecter. Physiology of the      Bacterial Cell: A Molecular Approach, Sinauer Associates, Sunderland, MA      (1990), pp. 351-388; Magasanik and Neidhardt in E. coli and Salmonella        typhimurium; Cellular and Molecular Biology (Neidhardt, F. C. et al. Eds.     pp. 1318-1325, American Society of Microbiology, Washington, DC (1987)!.      .sup.m use of limiting phosphate concentrations induces the phosphate         starvation regulon  Wanner in E. coli and Salmonella typhimurium; Cellula     and Molecular Biology (Neidhardt, F. C. et al. Eds., pp. 1326-1333,           American Society of Microbiology, Washington, DC (1987)!.                     .sup.n use of glutamine as a sole N source induces expression of the N        starvation regulon  Rietzer and Magasanik in E. coli and Salmonella           typhimurium; Cellular and Molecular Biology (Neidhardt, F. C. et al. Eds.     pp. 1302-1320, American Society of Microbiology, Washington, DC (1987)!. 

Example 11 Response of lon Transformed Host Cell to Ethanol or CopperSulfate

E. coli strain pLonE6/RFM443 was grown overnight at 26° C. in LB mediumcontaining Kanamycin (50 μg/mL) and diluted into the fresh LB mediumcontaining Kanamycin (50 μg/mL) and grown at 26° C. to early log-phase.50 μL of cells were added to 50 μL of LB medium containing Kanamycin (50μg/mL) and various concentrations of ethanol (added directly to themedium) or copper sulfate (diluted from a 250 mM stock solution inwater). Light output was quantitated as a function of incubation time ina Dynatech ML3000 luminometer at 26° C. The maximum response induced byethanol was observed when the final concentration of ethanol was 4%; at60 min after addition of ethanol the luminescence was 134 fold greaterin the presence of ethanol than in the untreated control. The maximumcopper sulfate induction resulted when the final concentration was 5 mM;at 60 min the induction ratio was 408 fold.

Example 12 Response of recA Transformed Host Cell to Mitomycin C,Ethidium Bromide or Cadmium Chloride

E. coli strains containing plasmid pRecALux3 were grown overnight at 26°C. in LB medium containing Kanamycin (50 μg/mL) and diluted into thefresh LB medium containing Kanamycin (50 μg/mL) and grown at 26° C. toearly log-phase. 50 μL of cells were added to 50 μL of LB mediumcontaining Kanamycin (50 gg/mL) and various concentrations of mitomycinC (diluted from a 2 mg/ml stock solution in water). Light output wasquantitated in a Dynatech ML3000 luminometer at 26° C. At 100 min afteraddition of 0.5 μg/mL mitomycin C, the induction ratios were as follows:

    ______________________________________                                        Strain DPD2791 (pRecALux3/W3110)                                                                            4.74                                            Strain DPD2794 (pRecALux3/RFM443)                                                                           20.00                                           Strain DPD2797 (pRecALux3/DE112)                                                                            15.70                                           ______________________________________                                    

E. coli strain DPD2794 demonstrated response to the presence of ethidiumbromide. Cells were grown overnight at 26° C. in LB medium containingkanamycin (25 μg/mL) and diluted into the fresh LB medium and grown at26° C. to early log-phase. 50 gL of cells were added to 50 gL of LBmedium containing various concentrations of ethidium bromide (dilutedfrom a 10 mg/mL stock solution in water). Light output was quantitatedin a Dynatech ML3000 luminometer at 26° C. At 180 min after addition of0.25 mg/mL ethidium bromide, the induction ratio was 1.9 fold.

E. coli strain DPD2794 was also shown to respond to the presence ofcadmium chloride by a disk diffusion assay. Cells were spread on an LBagar plate containing Kanamycin (50 μg/mL) and a filter disk which hadbeen wet with 20 gl of a 100 mM cadmium chloride solution was placed onthe agar plate. Following incubation overnight at 37° C., the agar platewas allowed to cool to room temperature. DuPont Reflection® film wasexposed to the plate for 10 min. Surrounding a zone of growth inhibition(18 mm diameter) a zone of enhanced bioluminescence (35 mm diameter) wasobserved.

Example 13 Response of KatG Transformed Host Cell to Methyl Viologen,Hydrogen Peroxide or Menadione

E. coli strains containing plasmids pKatGLux2 and pKatGLux6 were grownovernight at 37° C. in LB medium and diluted into fresh LB medium andgrown at 37° C. to early log-phase. 40 μL of cells were added to 60 μLof LB medium and various concentrations of methyl viologen (MV) whichwas diluted from a 200 mg/mL stock solution in water or hydrogenperoxide (H₂ O₂) which was diluted from a 0.3% solution in water. Lightoutput was quantitated in a Dynatech ML3000 luminometer at 26° C. Datais shown below in Tables VII and VIII.

                  TABLE VII                                                       ______________________________________                                        Strain  Time of Max Ind.                                                                           MV! for Max Ind.                                                                           Induction Ratio                             ______________________________________                                        DPD2507 60 min      8.75 mM*      56.7                                        DPD2508 60 min      8.75 mM*      19.5                                        DPD2509 60 min      8.75 mM*      31.9                                        DPD2510 80 min      8.75 mM*      11.2                                        DPD2511 60 min      8.75 mM*      226                                         DPD2512 50 min      2.2 mM        50.7                                        ______________________________________                                         *Maximum concentration tested.                                           

                  TABLE VIII                                                      ______________________________________                                        Strain  Time of Max Ind.                                                                           H.sub.2 O.sub.2 ! for Max Ind.                                                             Induction Ratio                             ______________________________________                                        DPD2507 65 min      1.65 mM       70.5                                        DPD2508 65 min      1.65 mM       3034                                        DPD2509 55 min      0.41 mM       250                                         DPD2510 50 min      0.41 mM       810                                         DPD2511 55 min      0.41 mM       376                                         DPD2512 45 min      0.41 mM       6067                                        ______________________________________                                    

E. coli strain DPD2511 was also shown to respond with increasedbioluminesence to the presence of menadione. Cells were grown overnightin LB medium containing kanamycin (25 μg/mL) at 26° C. and diluted to LBmedium and grown to log-phase at 26° C. 20 μL of cells were added towells of microtiter plates containing various concentrations ofmenadione (diluted from a 200 mg/mL solution in water) in 80 μL of LBmedium. Light output was quantitated in a Dynatech ML3000 luminometer at26° C. At 80 min the bioluminescence of cells treated with 2.3 mMmenadione was 1200-fold greater than in the untreated control.

Example 14 Response of MicF Transformed Host Cell to Methyl Viologen orHydrogen Peroxide

E. coli strains containing plasmids pMicFLux1 and pMicFLux2 were grownovernight at 37° C. in LB medium and diluted into the fresh LB mediumand grown at 37° C. to early log-phase. 40 μL of cells were added to 60μL of LB medium and various concentrations of methyl viologen (dilutedfrom a 200 mg/mL stock solution in hydrogen hydrogen peroxide (dilutedfrom a 0.3% solution in water). Light output was quantitated in aDynatech ML3000 luminometer at 26° C. Data is shown below in Tables IXand X.

                  TABLE IX                                                        ______________________________________                                        Strain  Time of Max Ind.                                                                           MV! for Max Ind.                                                                          Induction Ratio                              ______________________________________                                        DPD2515 120     min#    2.2    mM    99.9                                     DPD2516 50      min     8.75   mM*   14.7                                     DPD2517 120     min#    2.2    mM    1.8                                      DPD2518 120     min#    2.2    mM    1.4                                      DPD2519 120     min#    2.2    mM    87.5                                     DPD2520 120     min#    2.2    mM    69.2                                     ______________________________________                                         #Longest induction time analyzed.                                             *Maximum concentration tested.                                           

                  TABLE X                                                         ______________________________________                                        Strain  Time of Max Ind.                                                                           H.sub.2 O.sub.2 ! for Max Ind.                                                            Induction Ratio                              ______________________________________                                        DPD2515 65 min      1.65 mM      47.5                                         DPD2516 40 min      0.41 mM      5035                                         DPD2517 80 min      1.65 mM      4.2                                          DPD2518 80 min      1.65 mM      1.6                                          DPD2519 60 min      1.65 mM      2.2                                          DPD2520 60 min      1.65 mM      2.6                                          ______________________________________                                    

Example 15 Response of UspA Transformed Host Cell to Ethanol or CopperSulfate

E. coli strain DE134 containing plasmid pUspALux.2 was grown overnightat 26° C. in LB medium containing Kanamycin (50 μg/mL) and diluted intothe fresh LB medium containing Kanamycin (50 μg/mL) and grown at 26° C.to early log-phase. 50 μL of cells were added to 50 μL of LB mediumcontaining Kanamycin (50 μg/mL) and various concentrations of ethanol(added directly to the medium) or copper sulfate (diluted from a 250 mMstock solution in water). Light output was quantitated in a DynatechML3000 luminometer at 26° C. The maximum response induced by ethanol wasobserved when the final concentration of ethanol was 4%; at 60 min afteraddition of ethanol the induction ratio was 148 fold. The maximum coppersulfate induction resulted when the final concentration was 5 mM; at 60min the induction ratio was 6.9 fold.

Example 16 Response of xthA Transformed Host Cell to Propionate, Acetateor Hydrogen Peroxide

E. coli strains containing plasmids with the xthA promoter fused to thelux operon were grown overnight at 26° C. in LB medium containingKanamycin (25 μg/mL) and diluted into the fresh LB medium containingKanamycin (25 μg/mL) and grown at 26° C. to early log-phase. 50 μL ofcells were added to 50 μL of LB medium containing Kanamycin (25 μg/mL)and various concentrations of acetate (diluted from a 2M stock solutionin water), propionate (diluted from a 2M stock solution in water), orhydrogen peroxide (diluted from a 30% stock solution in water). Lightoutput was quantitated in a Dynatech ML3000 luminometer at 26° C. At 9 hafter addition of 0.025% hydrogen peroxide to strain DPD2778, thebioluminescence was 70-fold greater than in the control with noaddition; at 1 d after addition of 100 mM acetate, the induction ratiowas 54; and at 3 d after addition of 100 mM propionate the responseratio was 207. For strain DPD2781, at 18 h after the addition of 0.05%hydrogen peroxide the bioluminescence was 660-fold greater than in thecontrol with no addition; at 1 day after addition of 100 mM acetate, theinduction ratio was 61; and at 3 d after addition of 100 mM propionatethe induction ratio was 291.

Example 17 Response of His Transformed Host Cell to Gene Expression

E. coli strains containing plasmids with the his promoter fused to thelux operon were shown to be regulated by the stringent response systemin a genetic experiment showing the dependence of gene expression on thepresence of the appropriate regulatory elements. Plasmid DNA was placedby CaCl₂ mediated transformation into otherwise isogenic strains, withnormal regulation (strain CF1648), mutated in the relA regulatory gene(strain CF1693), or mutated in both the relA and spot regulatory genes(strain 1651). These strains were obtained from M. Cashel (Xiao et al.(1991) Residual Guanosine 3'5'-bispyrophosphate synthetic activity ofrelA null mutants can be eliminated by spoT null mutations. J. Biol.Chem., 266: 5980-5990). The strains were grown in LB medium containingAmpicillin (150 μg/mL) overnight at 37° C. and diluted in the samemedium and grown at 37° C. until early log-phase. Luminescence wasquantitated in a Dynatech ML3000 luminometer at 26° C. Three plasmidseach demonstrated reduced bioluminesence in a relA mutant, anddramatically reduced bioluminesence in the relA, spot double mutant.Data is shown below in Table XII.

                  TABLE XII                                                       ______________________________________                                        Plasmid  Host strain genotype                                                                       Fold reduction in bioluminescence                       ______________________________________                                        pHisLux5 relA.sup.+  spoT.sup.+                                                                      1X                                                     pHisLux5 relA.sup.-  spoT.sup.+                                                                      5.7X                                                   pHisLux5 relA.sup.-  spoT.sup.-                                                                     137X                                                    pHisLux9 relA.sup.-  spoT.sup.+                                                                      1X                                                     pHisLux9 relA.sup.-  spoT.sup.+                                                                      4.4X                                                   pHisLux9 relA.sup.-  spoT.sup.-                                                                     351X                                                    pHisLux12                                                                              relA.sup.+  spoT.sup.+                                                                      1X                                                     pHisLux12                                                                              relA.sup.-  spoT.sup.+                                                                      4.5X                                                   pHisLux12                                                                              relA.sup.-  spoT.sup.-                                                                     196X                                                    ______________________________________                                    

These fusions could also be induced by exposure to serine hydroxamate.This compound is a specific inhibitor of tRNA^(ser) aminoacylation byseryl-tRNA synthetase Pizer and Tosa (1971) J. Bacteriol. 106:972-982!.Cells were grown overnight with shaking at 29° C. in minimal E medium(Davis et al., Advanced Bacterial Genetics, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1980) supplemented with uracil (25μg/mL), kanamycin sulfate (10 μg/mL) and glucose (0.4%). The cells werediluted 20 fold in the same medium modified only by omission ofkanamycin sulfate and grown as noted above to between 19 and 34 KlettUnits. A 2 mg/mL solution of D,L-serine hydroxamate in water was diluted(serial 2-fold dilutions) in the same medium modified only by omissionof kanamycin sulfate. These dilutions (50 μL) were mixed with 50 μL ofactively growing cultures in a microtiter plate. Light output wasquantitated in a Dynatech ML3000 luminometer at 26° C. After 1180 min ofincubation the following induction ratios were observed:

                  TABLE XIII                                                      ______________________________________                                                               Ser. Hyd.!                                             Strain  Description   for Max Ind.                                                                             Induction Ratio                              ______________________________________                                        DPD1534 pHisLux5/RFM443                                                                             250 μg/mL                                                                             >2000                                        DPD1535 pHisLux9/RFM443                                                                             250 μg/mL                                                                             >1000                                        DPD1536 pHisLux12/RFM443                                                                            250 μg/mL                                                                             >400                                         DPD1540 pHisLux5/DE112                                                                              250 μg/mL                                                                             9                                            DPD1541 pHisLux9/DE112                                                                              250 μg/mL                                                                             3                                            DPD1542 pHisLux12/DE112                                                                             250 μg/mL                                                                             1                                            ______________________________________                                    

This data implies that we have capitalized upon the knowledge of thestringent response mechanism to develop a biosensor capable of detectinga wide range of amino acid biosynthetic inhibitors. Since manyherbicides are inhibitors of amino acid biosynthesis, this biosensor maybe a useful detector of several herbicides e.g., acetolactatesynthase-directed herbicides (including those in the sulfonylurea,imidazolinone, and triazolo- pyrimidine classes), phosphinothricin andglyphosate!.

Example 18 Response of phoA Transformed Host Cell to Limiting Phosphate

E. coli strains DPD1522 through DPD1533 containing plasmids with thephoA promoter fused to the lux operon were shown to respond to limitingphosphate. These strains were streaked for single colonies on MOPS media(Bochner et al. (1982) Complete analysis of cellular nucleotides bytwo-dimensional thin layer chromatography, J. Biol. Chem. 257:9759-9769) lacking tricine and containing glucose (0.4%) as the carbonsource, vitamin B1 (0.00002%), and a standard concentration of phosphate(2.0 mM) or a limiting concentration of phosphate (0.1 mM). Followingovernight incubation at 37° C., the plates were allowed to cool to roomtemperature and were exposed to DuPont Reflections® film for variousamounts of time. The strains growing on the standard concentration ofphosphate required 3 h to result in significant exposure of the film. Incontrast, the strains growing on limiting phosphate media required only1 min to yield significant exposure of the film, thus demonstratinginduction of bioluminesence by a limiting phosphate source.

This result was confirmed by an experiment conducted with a luminometer.Cultures were grown overnight with shaking at 29° C. in the aboveminimal medium containing 2 mM potassium phosphate supplemented withglucose (0.4%) uracil (25 μg/mL) and kanamycin sulfate (10 μg/mL). Cellswere collected by centrifugation prior to resuspension in an equalvolume of the same medium modified only by the omission of kanamycinsulfate and potassium phosphate. 50 μL of cells were added to 50 μL ofthe same medium lacking kanamycin sulfate and modified to give a finalconcentrations of potassium phosphate that ranged from 0-2000 uM. Lightoutput was quantitated in a Dynatech ML3000 luminometer at 26° C. as afunction of time for more than 300 min after addition of the resuspendedcells. Typical results for two strains DPD1522 (pPhoALux3/W3110) andDPD1523 (pPhoALux4/W3110)! are presented below:

                  TABLE XIV                                                       ______________________________________                                                            Maximal     Initial Induction                             Strain  uM Phosphate                                                                              Induction Ratio                                                                           Time (min)*                                   ______________________________________                                        DPD1522  0          1000         20                                                    31         500          50                                                    63         400         180                                                   125          3          260                                                   250         ni          ni                                                    500         ni          ni                                                    1000        ni          ni                                                    2000        ni          ni                                            DPD1523  0          >900         20                                                    31         700          20                                                    63         400         125                                                   125         350         175                                                   250         ni          ni                                                    500         ni          ni                                                    1000        ni          ni                                                    2000        ni          ni                                            ______________________________________                                         ni: inducation not observed                                                   *time of measurable increase in luminesence over baseline reading        

Example 19 Response of glnA Transformed Host Cell to Glutamine as a SoleNitrogen Source

E. coli strain DPD2831 was grown overnight in minimal phosphate medium(Bender et al., (1977) Biochemical parameters of glutamine synthetasefrom Klebsiella aerogenes, J. Bacteriol, 129: 1001-1009) containing 0.1%(NH₄)₂ SO₄. These cultures were collected by centrifugation andresuspended in either the same medium (control) or in that mediumlacking (NH₄)₂ SO₄, but containing 0.004% glutamine as the sole nitrogensource. Luminescence was quantitated in a Dynatech ML3000 luminometer at26° C. At 62 min after resuspension the cells in the medium with thepoor nitrogen source (glutamine) had 62-fold greater bioluminesence thandid the control culture.

Example 20 Construction of lac Containing Plasmids and Host Cells

E. coli strains were constructed such that plasmid-borne lux genes ofVibrio fischeri were under control of the E. coli lac promoter. A 232basepair Pvu II to Eco RI fragment of pUC19 (Yanisch-Perron et al.,(1985) Improved M13 phage cloning vectors and host strains: nucleotidesequences of the M13mp18 and pUC19 vectors, Gene 33: 103-119) wasligated into Sma I and EcoRI digested pUCD615 (Rogowsky et al., (1987)Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58,J. Bacteriol, 169: 5101-5112) to yield pLacLux. This plasmid wasoriginally isolated in E. coli strain XL1-Blue (Bullock et al. (1987),XL1-Blue: A high efficiency plasmid transforming recA Escherichia colistrain with beta-galactosidase selection, Biotechniques, 4: 376-379)which contains an F'lacI^(q), so that the genes were inducible by IPTG.The plasmid was also placed by CaCl₂ mediated transformation into E.coli strains W3110, RFM443, and DEll2 (see Table V, Example 10).

Example 21 Response of lac Transformed Host Cell to Carbon Source Levels

Glucose is the preferred carbon source for E. coli. E. coli strainsTV1058 and TV1068 each containing the plasmid pLacLux were grownovernight in LB medium containing kanamycin (25 μg/mL) either lacking orcontaining 0.4% glucose at 26° C. The overnight cultures were dilutedand grown to early log phase in the same media as the overnight culture.Culture turbidity was measured with a Klett-Summerson colorimeter with a#66 red filter. Luminescence present in 50 μL of cell culture wasquantitated in a Dynatech ML3000 luminometer at 26° C. Data are givenbelow in Table XIV.

                  TABLE XV                                                        ______________________________________                                        Strain   Media      Klett Units                                                                             RLU/50 μL cells                              ______________________________________                                        TV1058   -glucose   18        32.4                                            TV1058   +glucose   22        0.087                                           TV1068   -glucose   21        29.1                                            TV1068   +glucose   21        0.084                                           ______________________________________                                    

Thus, cells containing the plasmid pLacLux increase bioluminescence whengrown on the suboptimal carbon sources present in LB medium.

Example 22 Construction and Response of fabA Transformed Host Cell toFatty Acid Starvation

The fabA gene encodes the enzyme responsible for the placement of adouble bond in the fatty acids and hence membrane of E. coli. Suchdouble bonds are an absolute requirement for growth. Synthesis of fabAis directed by two promoter elements: a low level, constitutive upstreamand an inducible downstream promoter. The location of the two promotersin the sequence surrounding fabA has been determined. The PCR primersshown in Table IV are designed to allow cloning of the inducibledownstream promoter without the constitutive upstream promoter. Thetranscription of the downstream promoter can be modulated at least 10fold at the RNA level (Henry et al., J. Mol. Biol. 222:843-849 (1991)).Control of the dual fabA promoters, studied in fabA-lac fusions, hasshown a 13 fold modulation at the level of β-galactosidase specificactivity (Henry et al., Cell, 70: 671-679 (1992)). Control of fabAexpression is mediated by the fadR gene product (Nunn et al., J.Bacteriol, 154:554-560 (1983)). The FadR protein stimulates fabAtranscription by binding to the -40 region of the regulated, downstreamfabA promoter. If there is an excess of membrane synthetic capacity,long-chained acyl-CoA molecules accumulate. These molecules bind to theFadR protein, dissociating it from the regulated fabA promoter (Henry etal., Cell, 70: 671-679 (1992)). The fabA-lux fusion is thus expected toserve as a monitor of the state of membrane synthesis. Under conditionsof fatty acid starvation, long chain acyl-CoA pools will be low andexpression of fabA-lux should be high; excess fatty acids should resultin large pools of long chain acyl-CoAs and hence low levels of luxexpression from the fusion. This fusion should monitor not only fattyacid synthetic inhibition but CoA availability, which can be limited bymany factors including inhibition of the isoleucine-valine syntheticenzyme acetolactate synthase (LaRossa et al., pp. 108-121 inBiosynthesis of Branched Chain Amino Acids, ed. by Barak, Chipman andSchloss, VCH Publishers, New York, 1990). Methods of producing thestress of fatty acid starvation on a potential detector orgasmcontaining a fabA::lux fusion might include inhibition of fatty aciddesaturation by inclusion of 3-decenoyl-N-acetylcysteamine in the growthmedium (Nunn et al., (1983) J. Bacterlol, 154:554-560) or thesequestering of intracellular CoA as propionyl-CoA by the action of theherbicides such as sulfometuron methyl (Van Dyk et al., Mol Gen Genet(1987) 207:435-440) or the amino acid valine (LaRossa et al., (1987) J.Bacteriol, 169:1372-1378).

Construction of fabALux and Transformation of RFM443

Construction of a transforming plasmid containing the fabA::lux genefusion is prepared using methods and materials essentially as describedin Example 3 for the preparation of pRY001 and pRY002. PCR amplificationof the fabA promoter is accomplished with the primers listed in TableIV. The sequence of the fabA gene is known and is readily available fromthe Genbank database of nucleic acid sequences. The plasmid carrying thefabA::lux fusion is referred to as pFabALux.

E. coli host RFM443 is transformed with the pFabALux in using thematerials and methods described for the construction of WM1021 andWM1202 in Example 3.

E. coli host RFM443 is transformed with the pFabALux is grown overnightin minimal E medium containing kanamycin (10 μg/mL) at 29° C. Theovernight cultures are diluted and grown to early log phase in the samemedia as the overnight culture. Culture turbidity is measured with aKlett-Summerson colorimeter with a #66 red filter. Luminescence presentin 50 μL of cell culture in the presence or absence of 50 μg/mL ofsulfometuron methyl is quantitated in a Dynatech ML3000 luminometer at26° C. It is seen that cultures in the presence of sulfometuron methyldemonstrate a 10-25 fold increase in luminesces when compared withcultures in the absence of sulfometuron methyl.

Thus, cells containing the plasmid pFabALux are expected to increasebioluminescence when grown under conditions of fatty acid synthesisinhibition.

Example 23 Responses to a Physical Challenge

The recA::lux, uvrA:lux, grpE::lux, dnaK::lux, katG::lux, micF::lux anduspA::lux fusions were exposed to ultraviolet light irradiation. All butgrpE::lux responded to this physical challenge by increasingbioluminescence. Strains were grown overnight with shaking in LB mediumsupplemented with kanamycin sulfate (25 μg/mL) at 26° C. After 20-folddilution into LB medium, culture was continued with shaking at 26° C.until densities of 20-40 Klett units were reached. Cultures (50 μl) andfresh LB medium (50 μl) were added to wells of a microtiter plate priorto irradiation at 254 nm with a Stratalinker 1800 instrument(Stratagene). Subsequently, light output was quantitated as a functionof time after irradiation in a Dynatech ML3000 luminometer at 26° C.Response ratios were calculated after 240 min of incubation. They arereported in the Table XVI:

                                      TABLE XVI                                   __________________________________________________________________________    Response Ratio:                                                                     DPD2794                                                                             DPD2815                                                                             TV1061                                                                              WM1202   DPD2507                                                                             DPD2515                                                                             DE158                            Dose  (pRecAlux3/                                                                         pUvrALux2/                                                                          (pGrpElux.5/                                                                         pRY002/RFM443                                                                         (pKatGLux2/                                                                         (pMicFLux1/                                                                         (pUspALux13/                     (mjoule/cm.sup.2)                                                                   RFM443)                                                                             W3110 RFM443)                                                                             dnaK fusion)!                                                                          W3110)                                                                              W3110)                                                                              RFM443)                          __________________________________________________________________________    0     =1    =1    =1    =1       =1    =1    =1                               0.1    1     1    1      1       1     1     1                                0.4    2     2    1      2       1     1     1                                2      5    14    1.2    5       1.2   1.5   2                                10    10    29    1.5   20       2     4     2.5                              50    15    57    1.5   30       2.5   7     3                                250   15    63    <1    25       3     10    5                                1250  19    76    <1    20       2.5   10    8                                __________________________________________________________________________

It is apparent that all but one of the tested constructs responded tophysical as well as chemical stresses.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 24                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTTAGCGGATCCAAAAGCACAAAAAAT27                                                 (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AGCAGTGAATTCCATCTAAACGTCTCCA28                                                (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ACTTAAGGATCCAAGCGATGGCGCGTAAAA30                                              (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AGCAGCGAATTCATCGCCGCTTCCAGACAA30                                              (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ACTTAAGGATCCAGAGAAGCCTGTCGGCAC30                                              (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AGCTTTGAATTCCGCTTCTGTTTGTTTT28                                                (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ACTTTTGGATCCGTGTAAACGCGCGATTG29                                               (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AGCAGCGAATTCTTCCCGGATTAAACGCTT30                                              (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ACTTAAGGATCCCGAAATGAGGGCGGGAAA30                                              (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AGCAGCGAATTCGAACGTTGCTGACCACGA30                                              (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ACTTAAGGATCCCCCCAAAAATGCAGAATA30                                              (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AGCAGCGAATTCGGGCATCCGGTTGAAATAG31                                             (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ACTTAAGGATCCGCCATTACGTTGGCTGAA30                                              (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AGCAGCGAATTCCCACCCGTTTCGGTCATT30                                              (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ACTTAAGGATCCCTCCCGATACGCTGCCA29                                               (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AGCAGCGAATTCGGCGATGAGAATGTGTTTAT32                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ACTTAAGGATCCAATTACTGCGCCATTCTG30                                              (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ACATCGGAATTCTCATAGTCGCTGCCATTT30                                              (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      ACTTAAGGATCCCTAATTGTACGCATGTCA30                                              (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AGCAGCGAATTCAAAGTCTCTGTGAATGTT30                                              (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      ACTTAAGGATCCAGATTATCGTCACTGCAA30                                              (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AGCAGCGAATTCGGCCAATCAGCAAAATAA30                                              (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      ACTTTCGGATCCTTGGTGCAACATTCACAT30                                              (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AGCAGCGAATTCTCAGCGGACATCGTCAGT30                                              __________________________________________________________________________

We claim:
 1. A method of detecting the presence of an environmentalinsult comprising:(a) exposing a transformed detector E. coli to anenvironmental insult, the transformed detector E. coli being geneticallyengineered to contain an expressible heterologous luxCDABE gene complexunder the control of a stress-inducible promoter sequence wherein thepromoter sequence is responsive to a regulatory circuit; and (b)measuring an increase in luminescence of the transformed detector E.coli, the increase indicating the presence of an environmental insult.2. A method of detecting stress in a population of transformed E. colicomprising:(a) exposing a population of transformed detector E. coli toan environmental insult, the transformed detector E. coli beinggenetically engineered to contain an expressible heterologous luxCDABEgene complex trader the control of a stress-inducible promoter sequencewherein the promoter sequence is responsive to a global regulatorycircuit; and b) measuring an increase in luminescence of the transformeddetector E. coli, the increase indicating stress.
 3. The method of claim1 or 2 further comprising correlating the increase in luminescence tothe level of environmental insult present.
 4. The method of claim 1 or 2whereto the environmental insult is sublethal.
 5. The method of claim 1or 2 wherein the environmental insult is selected from the groupconsisting of atrazine, benzene, copper sulfate, 2,4-dichlorophenoxyacetic acid, ethanol, methanol, 2-nitrophenol, 4-nitrophenol,pentachlorophenol, phenol, toluene, dimethylsulfoxide, lead nitrate,cadmium chloride, sodium chloride, menadione, ethidium bromide, serinehydroxamate, acetate, propionate, hydrogen peroxide, puromycin, mercurychloride, 2,4-dichloroaniline, propanol, butanol, isopropanol, methylenechloride, Triton X100, acrylamide, methyl viologen, mitomycin C, xyleneand ultraviolet irradiation.
 6. The method of claim 1 or 2 wherein theenvironmental result stimulates a stress response selected from thegroup consisting of:(i) protein damage time alters the action of rpoH orits gene product, (ii) oxidative damage that alters the action of oxyRor its gene product, (iii) oxidative damage that alters the action ofsoxRS or their respective gene products, (iv) membrane damage thatalters the action of fadR or its gene product, (v) amine acid starvationthat alters the action relA and spoT or their respective gene products,(vi) carbon starvation that alters the action of cya and crp or theirrespective gene products, (vii) phosphate starvation that alters theaction of phoB, phoM, phoR, and phoU or their respective gene products;(viii) nitrogen starvation that alters the action of glnB, glaD, glnG,and glnL or their respective gene products; (ix) the universal stressresponse that alters the action of its regulators; (x) the stationaryphase response that alters the action of rpoS or its gene product; and(xi) DNA damage that alters the action of lexA or recA or theirrespective gene products.
 7. The method of claim 1 or 2 wherein thetransformed detector E. coli is in the log phase when exposed to theenvironmental insult.
 8. A transformed bioluminescent E coli capable ofan increase in bioluminescence upon exposure to a sublethal level ofenvironmental insult, the transformed bioluminescent E. colicomprising:(a) a stress inducible promoter sequence wherein the promotersequence is responsive to a regulatory circuit; and (b) an expressibleheterologous luxCDABE gone complex under the control the stressinducible promoter sequence.
 9. The transformed bioluminescent E. coliof claim 8 further comprising a tolC⁻ mutation wherein the mutationalters the permeability of the cell envelope of the E. coli to ahydrophobic environmental insult.
 10. A method of detecting the presenceof a environmental insult comprising:(a) exposing a transformed detectorE. coli to a sublethal environmental insult, the transformed detector E.coli being genetically engineered to contain an expressible heterologousluxCDABE gone complex under the control of a stress-inducible promotersequence wherein the promoter sequence is responsive to a regulatorycircuit; and (b) measuring an increase in luminescence of thetransformed detector E. coli, the increase indicating the presence of anenvironmental insult.
 11. E. coli selected from the group consistingof:(i) TV1076 having ATCC Number 69314 comprising a tolC⁻ mutation andan expressible heterologous lux gene complex under the control of a grpEstress inducible promoter sequence; (ii) WM1302 having ATCC Number 69316comprising a tolC⁻ mutation and an expressible heterologous lux genecomplex under the control of a dnaK stress inducible promoter sequence;(iii) TV1060 having ATCC Number 69142 comprising an expressibleheterologous lux gene complex under control of a grpE stress induciblepromoter sequence; (iv) TV1061 having ATCC Number 69315 comprising anexpressible heterologous lux gene complex under control of a grpE stressinducible promoter sequence; (v) WM1021 having ATCC Number 69141comprising an expressible heterologous lux gene complex under control ofa dnaK stress inducible promoter sequence; (vi) WM1026 having ATCCNumber 69143 comprising an expressible heterologous lux gene complexunder control of a dnaK stress inducible promoter sequence; and (vii)WM1202 having ATCC Number 69313 comprising an expressible heterologouslux gene complex under control of a dnaK stress inducible promotersequence.
 12. A nucleic acid molecule, comprising:(a) a stress induciblepromoter sequence wherein said promoter sequence is responsive to aregulatory circuit; and (b) an expressible bacterial luxCDABE genecomplex under control of said promoter sequence.
 13. The nucleic acidmolecule according to claim 12 wherein the stress inducible promotersequence is selected from the group consisting of groEL, dnaK, grpE,phoA, glnA, lon, lysU, rpoD, clpB, clpP, uspA, katG, uvrA, frdA, micF,fabA, lac, his, sodA, sodB, soi-28, recA, xthA, and narG.